EFFECT OF COLOSTOMY ON EXPERIMENTALLY PRODUCED NEOPLASMS OF T H E COLON OF T H E RAT

ANTONIO NAVARRETE, MD,” AND HARLAN J. SPJUT, MD

As a means of determining the effect of the fecal stream in the pathogenesis of experimentally produced colonic tumors in Wistar strain rats, simple colos- tomies were performed in the left colon of 57 female rats a t approximately 4 cm above the anus. Fifty-four rats received daily subcutaneous injections, 5 days per week, of the carcinogen 3:2’dimethyl-4-aminodiphenyl at a dosage of 2 mg/day/100 Gm body weight. Of the 54 rats, 17 (31.4%) developed either hy- perplasia, adenomatous polyps or carcinoma of the large bowel proximal to the colostomy site. None of the 54 rats developed any lesion distal to the colostomy. None of the three control rats with colostomies developed any kind of tumor. Of the colonic lesions that developed, 12 were hyperplasias, 12 were adenom- atom polyps and four were carcinomas. All hyperplasia developed at colos- tomy edge. Diversion of the fecal stream prevents development of colonic neo- plasms in Wistar rats in this experimental situation.

HEMICAL CARCINOGENS HAVE PROVIDED A C valuable tool for the experimental pro- duction of tumors in animals. Tumors of the bowel, particularly colon, have been success- fully induced in animals in several ways,l, lo, l2

mainly by the use of the chemical carcinogens of the aromatic amines group in Wistar strain rats.91 13, 14,10-l8 Thus, a successful experimen- tal model has been developed that makes pos- sible a study of the morphology, growth, dis- tribution and fate of colonic tumors in rats. In a previous experiment in our laboratory14 colonic adenomatous polyps and carcinomas were induced in 32% of white Wistar rats by the use of 3-2’dimethyl-4-aminodiphenyl, the majority of the tumors appearing in the dis- tal 4 cm of the large bowel.

Probably there are several factors attendant in the genesis of the induced colonic tumors. The role of fecal material containing the car- cinogen has been considered to be of possible importance and is the subject of the present experiment. That the fecal stream is of some importance in regression or disappearance of

From the Department of Pathology, Baylor Univer- sity College of Medicine, Houston, Texas 77025.

Supported by grant Ca 06828 from the National In- stitutes of Health.

*Work done while a FeIlow of the American Can- cer Society, Texas Division.

Address for reprints: Antonio Navarrete, MD, De- partment of Pathology, Baylor University College of Medicine, Houston, Texas 77025.

Received for publication November 4, 1966.

adenomatous polyps and carcinomas of the rectum and colon in humans has been well documented. This regression has followed co- lostomy in the case of carcinoma5-7 and sub- total colectomy and ileocolostomy in the case of familial polyposis.3~ 4, 6 , 8, 11

METHODS

Simple colostomies were created in the left colon of 57 female Wistar rats at 3 to 5 cm above the anus. Fifty-four of the 57 rats re- ceived daily subcutaneous injections, 5 days per week, of the carcinogen 3-2’dimethyl-4- aminobiphenyl in USP peanut oil a t a dosage of 2 mg/day/100 Gm body weight. T h e injec- tions were given in the flank region, alternat- ing sides from day to day. Rats that appeared ill or lost weight were not given injections for short periods until they began to gain weight or appeared healthy again. Mammary tumors were removed as they developed to prolong the life of the animal. The other three rats with a colostomy served as controls and received an equivalent dose of peanut oil. Two other rats without colostomies re- ceived normal diet only and served as weight controls.

Dosing was usually started 4 to 7 days after the colostomy. At the start of the experiment the rats were 6 weeks old and each weighed about 90 Gm. The shortest time elapsed be- tween onset of treatment and the sacrifice or spontaneous death of the group of 54 rats

1466

No. 9 COLONIC NEOPLASMS OF RATS - Navarrete and Spjut 1467

was 109 and the longest 344 days. All animals were examined by autopsy. The lesions were localized in relation to their distance from the ileocecal valve and the colostomy site. The greatest dimensions of all of the bowel lesions and of the other tumors were meas- ured. Representative sections of all tumors and most organs were taken unless autolysis made sections useless. Tissue sections were stained with hematoxylin and eosin; step sec- tions of the bowel lesions were made when needed.

RESULTS

From the group of 54 rats that received the carcinogen, 28 died and 26 were sacrificed. Seventeen, 34 and three rats died or were sac- rificed in the time periods of 101-200, 201- 300, and more than 300 days, respectively. Twenty-four rats had pneumonia, 16 when they died and eight when they were killed. One of the three rats with colostomies that served as controls was killed 168 days after the beginning of the experiment because of pneumonia: the other two were killed 366 days after onset of treatment and had no ab- normalities at autopsy.

Of the 54 rats that received the carcinogen, 42 (77.7y0) developed one or more neoplasms of some organ and only one of the tumors developed at the site of injections. Mammary

carcinomas were the most frequent tumors, occurring in 26 rats. Seventeen (31.4%) of the 54 rats developed either hyperplasia, adeno- matous polyps or carcinoma of the large bowel proximal to the colostomy site (Table 1, Fig. 1, 2). None of the 54 rats developed any le- sion of the large bowel distal to the colostomy site. None of the three control rats with colos- tomies developed any kind of tumor. Among other tumors that developed in the group of 54 rats that received the carcinogen were va- ginal, gastric, and soft tissue sarcoma; small bowel adenocarcinoma; bladder papilloma, xanthofibroma; lymphoma; sebaceous-squa- mous cell carcinoma of the ear and sarcoma of salivary gland.

The first extra-colonic tumors appeared 2y2 months after the first dose of the carcinogen; they were mammary carcinomas and were smaller than 1 cm when first detected. The earliest colonic lesion discovered was hyper- plasia of the colon mucosa on the lip of the colostomy site in an animal that was killed 131 days after the dosing began and had se- vere pneumonia with abscess formation.

Two of 17, 12 of 34, and three of three rats that died or were sacrificed in the time pe- riods of 101-200, 201-300, and more than 301 days, respectively, developed colonic lesions. The ratios (1 to 8.5; 1 to 2.6; 1 to 1) suggest that the longer the animals lived the greater the chances of development of colonic and

TABLE 1. Summary of Results

Total dose Animal carcinogen Total Type of Site in

no. (mg/100 ml) days lesion colon* Other tumors

4a

5a 14b 15a 15b

17a 17b 18a 22a 22b 23a 23b 24a 24b

2 6a

26b 27b

577.8

666.4 452.4 378.4 800.4

477.2 402.8 674.4 550.8

690.8 710.8 620.0 591.6

704.4

486.4

346.8 262.4

217

261 192 213 2 62

31.5 ~~.

219 279 238 267 340 344 203 259

250

265 131

H# and P

H and P H and Ca P Ca

H, P and Ca P H and P H and P P H and P H and P H H a n d P

Ca

H and P H

Edge of CS

3 . 5 from ICV 5 . 5 from CS 2.0 from ICV 2.3 from ICV

Edge of CS 4 from ICV 2.2 from ICV 3.2 from ICV 3.5 from CS Edge of CS Edge of CS

1.5 from CS

3.0 from ICV

Edge of CS

Ca, breast Sarcoma, abdominal cavity None Ca, breast (4) Ca, breast (2) Fibroadenoma Ca, breast Gastric sarcoma Sq. cell ca, ear None Ca, breast (3) None Sq. cell ca, ear Lymphoma Ca, breast (3) Sq. cell ca, ear Sarcoma, salivary gland Ca, breast (2) Papilloma, bladder Vaginal sarcoma None

Abbreviations: H-hyperplasia; P-polyp; Ca-carcinoma; ICV-ileocecal valve; CS--colostomy stoma. * Distance in centimeters of polyps and carcinoma in relation to the ileocecal valve and the colostomy. # All hyperplastic lesions were a t the stoma.

1468 CANCER September 1967 V O l . 20

other tumors. The dose of the carcinogen of the 17 rats that developed tumors varied greatly with a range of 262 to SO0 mg total dose. The dosage range for those that did not develop a tumor of the large bowel was 276 to 760 mg total dose (Table 1).

Seven of the 17 animals with colonic lesions had single growths: three had adenomatous polyps; two had carcinomas and two had hy- perplasia (Fig. 3-6). The other ten rats de- veloped multiple growths: eight had adenom- atous polyps and hyperplasia; one had a carcinoma plus hyperplasia; one had all three lesions. All 12 hyperplastic lesions of the colon developed at the edge of the colostomy

FIG. 1. Five adeno- matous polyps and one carcinoma of the colon proximal to co- lostomy stoma of a rat that received 477 mg carcinogen in 315 days. The carcinoma is the largest lesion seen near the stoma. T h e tumors ranged from 3 to 10 mm in greatest dimension (BtJCM 65320).

or within 1 cm proximal to it. None of three control rats developed hyperplasia of the colonic mucosa at any level.

Of the single growths, both carcinomas and two of the three polyps occurred in the proxi- mal colon within 3 cm of the cecum. Of the multiple growths, six occurred at the edge of the colostomy (five polyps associated with hy- perplasia and one neoplasm with all three lesions); three occurred within 3.5 cm of the cecum (polyps plus hyperplasia); one carci- noma was present midway between the cecum and the colostomy site. T h e dosages and dos- ing time for this group of animals is sum- marized in Table 1.

FIG. 2. Four colonic lesions at and near the colostomy stoma of a rat that received 591 mg of carcinogen in 259 days. Two were adenomatous polyps and two were hyper- plasia. The most prox- imal lesion represents the cut surface of an adenomatous polyp. The two lesions at the edge of the stoma are hyperplastic polyps.

No. 9 COLONIC NEOPLASMS OF RATS * Navarwte and Spju t 1469

FIG. 3. Adenomatous polyp of cecum of rat that received 402 mg of carcinogen in 219 days. The lesion meas- ured 3 mm in diam- eter (BUCM 18793-B; H and E, x21).

DISCUSSION tumors and exert little if any local carcino- genic action; the tumor was a xanthogranu-

Only one tumor occurred at the site of the loma which was probably secondary to the injection of the carcinogen in this experiment peanut oil used as the vehicle for the carcin- which is in agreement with previous observa- ogen. Whether the carcinogenic action is tions that the aromatic amines produce distant due only to the amine or to its metabolites or

FIG. 4. Focus of ade- nomatous epithelium present in colon of same rat as in Fig. 3. This lesion measured 1 mm in diameter and was present near the colostomy site. Two other smaller but sim- ilar areas of adenom- atom epithelium were present near this

and E, ~ 4 5 ) . (BUCM 18793-B: H

1470 CANCER September 1967 VOl. 20

to both is not known. Walpole, TVilliams and Roberts concluded that the effective carcino- gen was a metabolite of the amine given and that the appearance of tumors throughout the intestinal tract 01 Wistar rats suggested that the metabolites were excreted in the bile.16

If one postulates that only a metabolite(s) of the amine is the active carcinogen(s), it could be concluded that the obvious reason why the defunctionalized bowel did not de- velop tumors was because no feces containing carcinogen passed through it. If we postulate that the active carcinogen is only the given amine and not its metabolite, we would have to invoke other factors such as the “resting” of the defunctionalized bowel or decreased mitotic activity in the epithelium in protect- ing it from neoplasia since unquestionably the carcinogen also would reach the defunc- tionalized bow7el via the blood stream.

The occurrence of extra-intestinal tumors, in the present as in other experiments in which the same carcinogen and the same strain of rats have been used, indicates that its effect is to a great degree systemic rather than local, that it, that the carcinogen acted through the blood stream. It is possible that the amine as given, played a part in the development of the intestinal and extra-intestinal tumors; however, from our observations and those of

FIG. 5. Samc rat as in Fig. 1. Photomicro- g r a p h of a d e n o - carcinoma invading through the muscu- lark mucosa and into the submucosa (BUCM 18793-C; H and E, X26).

others16 it appears that a metabolite(s) ex- creted through the biliary system played a major role in the development of the intes- tinal tumors. Acting systemically, the metab- olite(s) were likely instrumental in the in- duction of the extra-intestinal tumors.

The results of this experiment also suggest that, under the circumstances presented, the fecal stream, perhaps acting as a “co-carcino- gen,” is an important factor in the genesis of the colonic neoplasms. Our results indicate that diversion of the fecal stream prevents the development of colonic neoplasms in Wistar rats in this experimental situation. Similar clinical situations seem to point in the same direction. Well-documented human cases with tissue diagnosis have indicated that diversion of the fecal stream may play a role in the regression or disappearance of carcinoma of the ~olon.~-7 In most of these instances irra- diation has been used in conjunction with the colostomies, making more difficult the evaluation of the role of diversion of the fecal stream per se. Dunphy reported a case of a 46-year-old man with carcinoma of the rec- tum which disappeared following a prelimi- nary colostomy without associated roentgen therapy.0

Regression or disappearance of polyps in the rectum of patients with familial polyposis following subtotal colectomy and ileocolos-

No. 9 COLONIC NEOPLASMS OF RATS - Nnvnnv te nnd Sp j i i t 147 1

FIG. 6. Hyperplasia of the colonic mucosa of a rat that received 620 mg of carcinogen in 203 days. Notice the thickness of the muscularis propria on the left to compare with the mucosal thickening (BUCM 18793-B; H and E, X21).

tomy is a more common phenomenon.3*4,6,8,11 Here also, in most of the instances, fulgura- tion has been used in conjunction with the surgical procedure. Whether adenomatous polyps regress as a result of colectomy or be- cause the ileal contents are discharged di- rectly into the retained rectal segment is not known. Several possibilities as the cause for disappearance of the polyps have been sug- gested:

1. Elimination of an intrinsic factor by removal of the bulk of the colonic mu- cosa which supposedly produces or acti- vates a carcinogenic 3, l5

2. Existence of a substance in the ileal contents which inhibits a growth-induc- ing stimulus believed to be present in the large bowel m u ~ o s a ; ~

3. Diminution in the blood supply to the remaining rectal ~egment .~

Of special interest is the case published by Cole et al.4 of a 34-year-old woman with famil- ial polyposis who underwent ileoproctostomy and total colectomy in two stages. Anastomosis of the proximal end of the divided ileum to the right lateral wall of the rectum at the level of the peritoneal reflexion was performed with the defunctionalized unremoved portion

of the colon remaining for 10 months. Spon- taneous regression of adenomatous polyps in the rectum, distal to the ileoproctostomy and immediately proximal to the anastomosis, oc- curred. The authors also believed that there was some suggestion that the adenomas in the defunctionalized portion of the bowel showed an accelerated rate of growth. Based on these obsservations they concluded that the ileal fluid contains growth-inhibiting proper- ties which may neutralize a growth-inducing stimulus believed to be present in the mucosa of the large bowel of patients with colonic polyposis.

CONCLUSIONS

The results of this experiment indicate that the diversion of the fecal stream prevents the development of colonic neoplasms in Wistar rats in this experimental situation. In the experimental circumstances presented, the passage of feces may be a factor, along with the carcinogen, in the genesis of the colonic neoplasms.

Possible future experiments that would be expected to add to our knowledge of the ac- tion of the carcinogen would be (1) radioac- tive tagging of the carcinogen to determine its localization; (2) diversion of the bile stream

1472 CANCER September 1967 V O l . 20

of animals receiving the carcinogen to deter- mine whether the colon will remain free of tumors; (3) feeding of other rats with the col- lected bile from animals receiving the carci- nogenic substance; (4) high colostomy in rats with established tumors, proximal to these tumors, to determine whether or not diver- sion of the fecal stream will produce regres-

sion of the tumors and (5) reanastomosis of the colon, after the colostomy has been estab- lished from 6 to 9 months, to determine whether tumors will appear in the previously defunctionalized bowel. The morphologJ of colonic lesions produced in the same strain rats and by the same carcinogen has been re- ported in several other papers.

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12. Nowell, p. c., Cole, L. ,,, and Ellis, M, E.: In- duction of intestinal carcinoma in the mouse by whole- body fast-neutron irradiation. Cancer Res. 16:873-876, 1956.

13. Spitz, S., Maguigan, W. H., and Dobiner, K.: The carcinogenic action of Benzidine. Cancer 3:789- 803, 1950.

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induced tumors of the intestine. ~ ~ ~ ~ t . N . zeal. J. surg. Bladder tumors induced in rats of two strains with 29~38-53, 1959. 3:2’dimethyl-4-aminodiphenyl. Brit. J. Cancer 9: 170-

10. Lisco, H. Brues, A. M., Finkel, M. P., and 1769 1955. Grundhauser, W.: Carcinoma of the colon in rats fol- 18. - , and Williams, M. H. C.: Aromatic amines lowing the feedings of radioactive yttrium. Cancer as carcinogens in industry. B i t . Med. Bul. 14:141-145, Res. 7:721, 1947. 1958.

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