Polyposis coli: An experimental animal model

Polyposis Coli: An Experimental Animal Model By R. Carachi Glasgow, S c o t l a n d 9 Patients w i t h familial polyposis, long standing ulcerative colitis and u r e t e r o s i g m o i d o s t o m i e s have an unstable colonic epithelium. T h e r e is a p r o p e n s i t y for p a t i e n t s w i t h t h e above conditions t o develop colonic dysplasia and neoplasia. An e x p e r i m e n t a l animal model using 1:2 d i m e t h y l h y d r a z i n e dihydrochloride (DMH) w a s used to induce dysplastic colonic lesions w i t h a potential for neoplastic g r o w t h . This model w a s used to t e s t t h e hypothesis t h a t by altering t h e colonic e n v i r o n m e n t one could change t h e natural h i s t o r y of t h e disease in this e x p e r i m e n tal model. In t h r e e separate e x p e r i m e n t s t o test this hypothesis, t h e first set d e m o n s t r a t e d t h e reliability of t h e model by inducing 8 to 10 t u m o r s in t h e colon of each one of 65 e x p e r i m e n t a l animals. These t u m o r s are v e r y similar in m o r p h o l o g y to polyposis coll. The second set of e x p e r i m e n t s involved alteration of t h e colonic e n v i r o n m e n t by having a group of rats on a high fiber diet. A t o t a l of 36 t u m o r s w e r e induced in t h e rats on a high fiber diet, w h e r e a s rats on a normal fiber diet had a t o t a l of 94 t u m o r s induced. The high fiber diet p r o t e c t s a dysplastic colonic mucosa and alters t h e natural history of t h e disease in this e x p e r i m e n t a l model (p value < 0.01 ). The t h i r d set of e x p e r i m e n t s w e r e designed in an attempt to identify the environmental factors responsible for t h e change in t h e course of t h e disease in rats on t h e high fiber diet. The fecal bile acid e x c r e t i o n , a k n o w n colon t u m o r p r o m o t e r , w a s significantly reduced in rats on a high fiber diet (p value < 0,01 ). The fecal residue w h i c h dilutes t o x i n s and binds carcinogens o n t o d i e t a r y fiber as an ion e x c h a n g e resin w a s f o u n d t o be significantly increased in t h e rats on a high fiber diet (p value < 0.01). The mean intestinal t r a n s i t t i m e w h i c h helps in t h e rapid elimination of any t o x i c w a s t e p r o d u c t s in t h e colonic e n v i r o n m e n t w a s significantly diminished in rats on t h e high fiber diet (p value < 0.05). The clinical relevance of this e x p e r imental model could be significant for pediatric patients w h o are predisposed t o colonic neoplasia. The course of this disease could be changed if t h e i r colonic e n v i r o n m e n t can be altered t o p r o t e c t t h e m from factors o p e r a t i n g on the unstable colonic epithelium. INDEX WORDS: Polyposis coil; fiber diet.

H E U N S T A B L E colonic epithelium in

T patients with familial polyposis, long standing ulcerative colitis, and ureterosigmoidostomy site is now well recognized. 1-3 There is a propensity for patients with the above conditions to develop colonic dysplasia and neoplasia. Mea-

Journal of Pediatric Surgery, Vol. 18, No. 1 (February), 1983

sures to reduce or eliminate this tendency are required. The three sets of experiments are based on the hypothesis that normal colonic epithelium is stimulated to undergo changes and become unstable. This then undergoes spontaneous changes to dysplasia, adenoma, adenocarcinoma and finally metastases. The first set of experiments is the development of a reliable model for polyposis coll. The hypothesis is tested in the second set of experiments by alteration of the colonic environment by a change in diet in an attempt to alter the natural history of the disease in the experimental model. The third set of experiments is the identification of the colonic environmental factors responsible for the change in the course of the disease in those rats on a high fiber diet. The factors investigated are the fecal bile acids which are known colon tumor promoters; the fecal residue, the lack of which has been incriminated in the high incidence of disease in western countries; the intestinal transit time, since toxic substances are in contact with colonic mucosa for a longer period if there is colonic stasis and could predispose to colonic cancer. E X P E R I M E N T A L PROCEDURES Subjects

The animals used in all three sets of experiments are male Sprague-Dawley rats with a starting weight of 200 g. The animals were fed on isocaloric diets (caloric value 11.21 megajoules/ kg diet) oxoid 41 without antibiotics. Those rats

From the Department of Surgical Paediatrics, Royal Hospital for Sick Children, Glasgow, Scotland. Presented before the XXIX Annual Congress of the British Association of Paediatric Surgeons, Madrid, Spain, July 21-23, 1982. Address reprint requests to R. Carachi, M.D., Department of Surgical Paediatrics, Royal Hospital for Sick Children, Yorkhill, Glasgow, G3 8S J, Scotland. This paper contains experimental work which is being submitted to Glasgow University and forms part of Ph.D. thesis. 9 1983 by Grune & Stratton, Inc. 0022-3468/83/1801--0012501.00/0

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52

on a normal diet had a low crude fiber content of 2.5%: rats on a high fiber diet had a crude fiber content of 15%. All the rats were injected subcutaneously with 1,2-dimethylhydrazine dihydrochloride ( D M H ) weekly at a dose of 15 m g / k g body weight. The control rats in all the experiments received a normal diet (oxoid 41 without antibiotics, crude fiber content 2.5%) and received injections with E D T A which was used as a carrier for stabilization of the carcinogen solution.

Experiment 1--Experimental Model Sixty-five male Sprague-Dawley rats were injected subcutaneously with D M H at a dose of 15 m g / k g body weight on a weekly basis till dysplastic changes were evident in the colon. Four rats were sacrificed weekly from the start of the experiment till dysplastic changes were present in the colon. The remaining rats were all sacrificed at 30 wk in the experiment. A full post mortem was carried out on each experimental animal and histopathology performed on all tissues and lesions induced. The control group of 20 animals received carrier substance E D T A and were sacrificed at similar time intervals to the experimental animals. The purpose of this first set of experiments was to determine the minim u m amount of carcinogen necessary to induce dysplastic areas in the colonic mucosa and minimise the toxicity caused by the carcinogen. The morphology of the tumors induced was assessed anatomically and morphologically.

Experiment 2--Dietary Fiber Three groups of rats were involved in this experiment: Group A - - 2 0 rats on an isocaloric normal diet (2.5% crude fiber) injected with D M H . Group B - - 2 0 rats on an isocaloric high fiber diet (15% crude fiber) injected with D M H .

R. CARACHI

Group C - - 2 0 control rats on a normal diet injected with carrier substance EDTA. All the rats were sacrificed for analysis of the tumors induced at 28 wk in the experiment.

Experiment 3--Environmental Factors Three groups of animals studied: Group A - - 1 0 rats on an isocaloric high fiber diet (15% crude fiber) similarly injected with DMH. Group B - - I 0 rats on an isocaloric normal diet (2.5% crude fiber) injected with D M H . Group C - - 1 0 control rats on a normal fiber diet injected with carrier substance EDTA.

Studies Measurements of the following were made: 1. Intestinal transit time was measured at 12 wk into the experiment. Under halothane anaesthesia 25 ball bearings, 1 m m in diameter were syringed down a nasogastric tube into the stomach. The tube was removed and on recovery the rats were fed their appropriate diet. Collection of feces over 4 hourly periods for 48 hr was made at the above times. The feces passed were x-rayed and the ball bearings counted. Transit time was the time taken for 50% of the ball bearings to appear after ingestion (Fig. 1). 2. Fecal residue was measured for weight and volume on the collected specimens described. 3. Fecal bile acid excretion was measured on fecal collections after extraction of fecal bile acids, measurements were made using the hydroxysteroid dehydrogenase enzyme assays. These were made on collections obtained from metabolic ages in which each rat was studied for a period of 24 hr on weeks 1, 8, 16, 20, 24 and 28. 4. Colonic lesions and metastases were detected at post mortem examination which was performed on all the rats when they were sacri-

Fig. 1. X-ray of a rat with ball bearings for measuring the mean intestinal transit time.

POLYPOSIS COLI: AN EXPERIMENTAL MODEL

53

riced at 28 wk from the start of the experiment. The tumors and metastases were identified, dissected out and weighed. Statistics The statistical test used is the Student's t test in all analyses.

RESULTS Experiment 1--Experimental Model The first set of experiments showed early multiple changes throughout the whole extent of the colon at four weeks. These changes on histological examination showed an increase in mitotic figures and increased basophilia in some of the crypts of the glands (Fig. 2). By 16 wk after weekly injections of the carcinogen small plaques were found on the colon, which showed areas of dysplasia on their surface. At this stage after 16 wk of injection of carcinogen, the injections were stopped and these lesions (8-10 per rat) were allowed to grow without any further carcinogenic stimulation. At 20 wk multiple syn-

Fig. 3.

Photograph of multiple polyps arising from the

descending colon of the rats on a normal low fiber diet.

chronous polypoidal tumors were found (Fig. 3) throughout the extent of the colon. The majority of tumors were well differentiated adenocarcinomas which eventually invaded the stalk of the polyp. By 26 wk the tumor had metastasised to the serosa and entered the lymphatics. At 28 wk the tumor had invaded the serosal boundary with metastatic spread. The transperitoneal spread with invasion of the diaphragm and the pleura was a terminal event (Fig. 4). Many of the experimental animals at this time were found to be suffering from the mechanical effects of this tumor causing tumor induced intussusception. The first set of experiments supported the hypothesis that a reliable experimental animal model had been established using 65 experimental animals with a constant induction of 8 to 10 tumors in the colon of each rat. These tumors were mainly left sided with a similar morphology to polyposis coli. Experiment 2 - - D i e t a r y Fiber

Fig. 2. Dysplastic changes in colonic mucosa induced by dimethylhydrazine dihydrochloride.

The second set of experiments involved an alteration of the colonic environment by the introduction of fiber in the diet. The rats on a high fiber diet had a total of 36 tumors induced: rats on a low fiber diet had a total of 94 tumors induced. These results were statistically significant (p < 0.01) (Fig. 5). An analysis of the tumors induced showed that there were four times as many adenomatous lesions in rats on a

54

R. CARACHI

Experiment 3 - - E n v i r o n m e n t a l Factors The intestinal transit time in Group A (rats on a high fiber diet) was 8 hr and in Group B and C, it was 12 hr. The decrease in intestinal transit time in the high fiber diet animals was significant

(p < 0.05). The fecal residue in Group A was significantly higher in volume ( p < 0.01) and weight (p < 0.01) compared to the normal fiber diet animals, groups B and C (Fig. 6). The total fecal bile acid excretion was significantly lower in Group A compared to groups B and C (p < 0.01) (Fig. 7). An analysis of the polypoidal tumors induced in the last set of experiments showed that a total of five polypoidal tumors were induced in rats on a high fiber diet, the total tumor weight was 1.4 g and 53 polypoidal a d e n o c a r c i n o m a s were induced in rats on a low fiber diet with a total tumor weight of 9.6 g. There were no tumors found in the control animals. These results were statistically significant (p < 0.01) (Fig. 8). DISCUSSION

Fig. 4. Colo-colic intussusception in a rat on a normal high fiber diet.

low fiber diet and three times as many polypoidal adenocarcinomas in rats on a low fiber diet. In addition tumors in the rats on a low fiber diet metastasised and two of these rats had evidence of liver metastases.

The incidence of spontaneous colon cancer in rats is 0.1%.4,5 The discovery of the carcinogenic affect of cycasine and its aglycone on intestinal mucosa by Lacqueur opened the way for research work on new synthetic intestinal carcinogens. 6 Druckery demonstrated that the synthetic dialkylhydrazines were organotropic for gastrointestinal mucosa. 7 The important considerations for developing a standard experimental

SITE OF COLORECTAL TUMOURS

Right Colon H.F.= (28%)10 L. F. : (19 ~o)18

i /"""

Left Colon H.F. : ( 6 4 ~ ) L,F. = ( 6 7 ~ )

23 64

Recto - S igmoid

Coecum H.F=O L.F =0

H.F. = ( 8 ~ ) 3 L.F. = ( 1 4 ~ ) 12

Fig. 5.

Diagrammatical

representation of macroscopic lesions induced by dimethylhydrazine in rats on a high fiber diet and on a normal low fiber

diet.

POLYPOSIS COLh AN EXPERIMENTAL MODEL

55

FAECAL RESIDUE Volume cc r 40. Group A -- High fibre Group B -- Low fibre Group C -- Control

30.

1

20-

'.'.1;.:.: =

,I

I i:i:i:!:i:i: :i:i:i:i:!:i .:.:,:,:.:,:

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A

B Groups

C

Fig. 6. Table illustrating the fecal bulk in the three groups of rats.

model include the type of carcinogen, the mode of administration, the dose and the species of animal used. Most of the carcinogens except for dimethylhydrazine and its metabolites azomethane and methylazoxymethane produce a low incidence of intestinal adenocarcinomas and the induction of extra intestinal tumors is common.8 In this model, a low dose of carcinogen was used which was sufficient to produce a fixed number of dysplastic areas in the colon in each rat. The withdrawal of t h e carcinogen then allowed these lesions to develop into adenomatous polyps and then carcinoma with a minimal side effect from drug toxicity. This model is a reliable and easily reproducible method for tumor induction within 20 wk. 2 The preponderance of polypoidal tumors arising from multiple loci of dysplasia bears some resemblance to patients with genetically unstable colonic mucosa who develop polyposis coll. The colonic mucosal environment is determined by the conditions prevailing intraluminally and experimental evidence in animals with colostomies show that they develop fewer tumors in the defunctioned bowel than they did in intact animals in the same bowel segment. 9 The results show that the fecal stream alters the carcinogenic activity of the compound in the large bowel of the rat. ~~ This model was used to study the effect of altering the colonic environment using an isocaloric high fiber diet. There was a significant decrease in the number of tumors induced in the rats on a high fiber diet. The preponderance of left sided lesions in rats who have tumors induced by this method can be explained by the increased concentration and duration of contact by the carcinogen on the DAILY FAECAL81LE ACIDEXCRETION

Collecti0n (weeks)

Fig. 7. Table illustrating total fecal bile acid estimation of rats on different diets and their significance.

0tet + O.M.H. High Fibre Low Fibre (GroupA) N = 10 (GroupB) N=10

0tet Control (GroupC) N=10

Statistical S19n|ffcance (P value)

14.0

28.0

23.1

A,B P
15.0

41.3

17.3

k,B P
1G

17.1

24.5

35.9

A,C P
20

19.1

31.7

25.5

A,B P
24

9.4

24.3

27.3

A,B P
28

9.5

26.3

22.3

A,B P
56

R. CARACHI

Groups

Lesion Plaque (dysplastic)

A (H.F.) ]0

B (N.F.) IO

c (c)]o

3

3

o

Sessile (adenomatous)

Polypold (adenocarcinoma)

@

Total Number Total Tumour Weight

53*

20

57

0

1.41296ga

9.65815gm

0

Fig. 8. Table illustrating types of tumors induced and their weights.

(A x B = P value
colonic mucosa. The protective role of the fiber in the diet may be due to a number of factors acting directly or indirectly affecting the intestinal transit time, the fecal bulk, and the fecal bile acid production. Intestinal Transit Time The transit time is the time taken for food residue to pass through the gut, and in humans this is shortened by the high fiber diet. ~ The more refined the diet, the smaller the stool and the slower the passage of the food residue through the intestine./2 It has been postulated that fiber may protect against colonic cancer by decreasing the intestinal transit time and so allow less time for environmental carcinogens to be in contact with colonic mucosaJ 3 Rats on a high fiber diet had a significantly reduced intestinal transit time compared to rats on a normal low fiber diet. Fecal Bulk, Fecal Weight, and Fecal Volume

The high dietary residue is one of the factors which is thought to affect the occurrence of large bowel cancer. 13 It may be protective against colonic carcinogens by its water absorbing properties where it increases the bulk of the stool and aids in propelling fecal residue as well as reducing transit time. It also increases volatile fatty acids which add to the fecal bulk and promotes a rapid transit time. The increase in bulk may help

to dilute the concentration of carcinogens in the colonic environment and may absorb carcinogen ions by acting as an ion-exchange resin deactivating them? 4 Rats on a high fiber diet had a greater fecal weight and fecal volume than rats on a normal diet. This was confirmed for measurements both before and after the tumors were induced. Fecal Bile Acids

Feces of people in areas with a high incidence of colon cancer have a high bile content and a highly anaerobic bacterial flora in the colon, capable of metabolizing endogenous bile acids to form endogenous intestinal carcinogens. 15 A number of bile acids have been demonstrated experimentally to be carcinogenic. 16'17Similarly an increase in the bile acid content of the large bowel by mechanical means has been reported to enhance the carcinogen activity of tumor induced in rats./8 The total fecal bile acid excretion was significantly reduced in rats on a high fiber diet, yet another mechanism that could be protecting them from developing neoplasia. The number of tumors induced in the rats on a high fiber diet was significantly smaller. A high fiber isocaloric diet in an experimental model for polyposis coli and colonic neoplasia induced by dimethylhydrazine protects the sites of dysplasia in the colonic mucosa.

POLYPOSIS COLI: AN EXPERIMENTAL MODEL

57

The m e c h a n i s m s outlined are: (1) The reduction of fecal bile acid excretion which acts as a colon t u m o r promoter in this model; (2) By increasing the total fecal bulk and diluting the carcinogen excreted in the bile; a n d (3) By the more rapid e l i m i n a t i o n of toxins a n d co-carcinogens through shortening the m e a n intestinal transit time that these substances have to come into contact with the colonic mucosa,

F u r t h e r support for this concept has now been recorded by other workers who have used this model for testing t u m o r induction at the sites of ureterosigmoidostomy. 3 This experimental model has clinical relevance to those pediatric patients with u n s t a b l e colonic epithelium and m a y benefit by this experi m e n t a l work in an a t t e m p t to change the p a t t e r n of their disease,

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9. Campbell RC, Singh DV, Nigro ND: Importance of the fecal stream on the induction of colon tumours by azoxymethane in rats. Cancer Res 35:1369-1371, 1975 10. Druckrey H: "Organospecific carcinogenesis in the digestivetract, in Nakahara W, Takayama S, Sugimura T, et al (eds): Topics in Chemical Carcinogenesis.Tokyo, University of Tokyo Press, 1972, pp 73-103 11. Cummings JH: Dietary Fibre. Progress Report, Gut 14:69-81, 1973 12. Walker ARP: Crude fiber, bowel motility and pattern of diet. South Afr Med J 35:114-115, 1961 13. Burkitt DP: Epidemiologyof cancer of the colon and rectum. Cancer 28:2-13, 1971 14. Fordtram .IS: Organic amines in faecal contents. N Engl J Med 284:329-330, 1971 15. Hill MJ, Drasar BS, Aries V, et al: Bacteria and etiology of cancer of the large bowel, Lancet 1:95-100, 1971 16. Wynder EL, Shigamatsu T: Environmental factors of cancer of the colon and rectum. Cancer 20:1520-1561, 1967 17. Lacassagne A, Buu-Hoi NP, Zajdela F: Carcinogenic activity of apocholic acid. Nature 190:1007-1008, 1961 18. Chomchai C, Bhadrachari N, Nigro ND: The effect of bile on the induction of experimental intestinal tumors in rats. Dis Colon Rect 17:310-312, 1974