Experimental colon cancer

001~5085/78/7506-1157$02.00/0 GASTROENTEROUJGY 75:1157-1169, 1978 Copyright 0 1978 by the American Gastroenterological Association

Vol. 75, No. 6 Printed in US’A.

PROGRESS EXPERIMENTAL

ARTICLE

COLON CANCER J. THOMAS

LAMONT

AND THOMAS

A.

O’GORMAN

Department of Medicine, Harvard Medical School, and the Division of Gastroenterology, H&-pital, Boston, Massachusetts

Cancer of the colon and rectum is second only to lung cancer as the commonest cause of cancer deaths in adult Americans. The disease, which causes approximately 50,000 deaths per year, is incurable in half of the patients at the time of initial diagnosis. Although the etiology of colon cancer is unknown, a number of epidemiological studies have suggested that environmental factors such as dietary fat may explain the higher incidence in urbanized Western society than in developing countries. ‘. Z However, these studies of human population groups provide very little information about the evolution and pathogenesis of colon cancer. In recent years a number of chemical carcinogens have been discovered which provide investigators with a reasonably accurate animal model of human colon cancer. These compounds when administered to rodents produce benign and malignant neoplasms of the colon which are strikingly similar in most respects to colon tumors in man. They provide the opportunity to study under controlled laboratory conditions the induction of colon cancer, and the effects of manipulation of the immune system, diet, gut bacteria, and other factors on tumor production. Our major objective is to review the available information (only studies published before January 1, 1978, are included in this review) regarding the chemistry, metabolism, and mechanism of action of the more widely used colonic carcinogens, and the biology of t.he tumors they produce.

Historical Background and Classification Carcinogens

of Colonic

Colonic carcinogens are defined as compounds which produce cancer of the large intestine; however, the majority also produce neoplasms in other organs. These compounds can be classified into five groups on the Received May 1, 1978. Accepted July 5, 1978. Address requests for reprints to: Dr. J. T. LaMont, Division of Gastroenterology. Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115. This study was supported by Clinical Investigator Award AM 00138 from the National Institutes of Health and a grant from the American Cancer Society. Dr. O’Gorman was supported by Training Grant T32-AM 07121 from the National Institutes of Health. The authors would like to thank Drs. Selwyn Broitman, Kevin V. Carey, Martin C. Carey, 2. Myron Falchuk, Steven C. Fiske, and Jerry S. Trier for their helpful criticisms and suggestions, and Ms. Teri Fox for her help in preparing the manuscript.

Peter Bent Brigham

basis of chemical structure (fig. 1). The first experimental induction of intestinal tumors was reported in 1941 by Lorenz and Stewart,3 who observed multiple small intestinal cancers but not colonic tumors in mice fed dibenzanthracene or methylcholanthrene. It was subsequently reported that feeding 3-methylcholanthrene produced colon cancers in males but not females of various strains of inbred hamsters.“ Although not widely used as experimental colonic carcinogens, cholanthrene derivatives are structurally quite similar to bile salts and cholesterol and, as noted below, may be produced within the human intestinal tract by bacterial metabolism of naturally occurring fecal steroids. The carcinogenicity of the substituted biphenyls, a by-product in the manufacture of dyes, was discovered in the early 1950’s by several groups investigating the high incidence of bladder cancer in dye industry workers. Walpole et al.” produced small intestinal and colonic adenocarcinomas in a small percentage of rats by subcutaneous injections of dimethylaminobiphenyl or 4aminobiphenyl. Compounds of this group are actively secreted in bile and then carried to the target cells via the fecal stream. Thus surgical removal of the colon from the fecal stream prevents colon tumors in rats treated with these carcinogens.“, i The serendipitous discovery in 1963 by Laqueur and his associates8 of the ability of cycad meal to cause colon cancer in rats led to the discovery that the hydrazine derivatives are potent colonic carcinogens. These investigators were searching for a possible dietary cause of the unusually high incidence of amyotrophic lateral sclerosis in Guam. They postulated that cycad meal made by grinding the nuts of the tropical plants Cycas circinalis might be neurotoxic to humans because animals which ate the leaves of the plant developed paralysis.” Furthermore, cycad flour was consumed in large amounts by native Guamanians. Feeding crude cycad meal to rats failed to produce neurological disease; however, adenocarcinomas of the colon were observed in a small percentage of animals. Laqueur very astutely recognized the potential importance of this unforeseen result, and subsequently demonstrated’“, ” that the carcinogen in crude cycad meal was cycasin, a watersoluble glucoside of methylazoxymethanol (fig. 2). The chemical instability of the latter compound’” and the difficulty in obtaining cycad nuts seriously limited the study of cycasin as a colonic carcinogen. These problems

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f.CHJLANTHRENES @

il

A#MAT/C

AMNES

h’ethykholonthrene

cckI.%b 2’. 3- Dtmethyl CH,mNHmN+CH,

3, HYDRAZ/NE L’XRIVA ,TIVES

1.2

4.

4 Amfnoblphenyl

Durethylhydrar~ne

AL WL NITTiOSAMlDES

5. AFLATOXIN

FIG. 1. Colon cancers have been produced in experimental animals with all of the above chemical carcinogens. The most potent and specific agent is 1,2_dimethylhydrazine.

CH,-NH+NHFC+

t CH,mN=N-CH,

+ Cti-N=N-CH,

6I

H&‘+N,

FIG. 2. Metabolic activation of dimethylhydrazine. 1,2-Dimethylhydrazine, azoxymethane, methylazoxymethanol, and cycasin are colonic carcinogens and are converted to the alkylating agent, methyldiazonium. This compound decomposes spontaneously to generate a highly reactive carbonium ion.

were overcome by Druckrey’s important discovery in 1967’:’ that 1,2_dimethylhydrazine selectively produced small intestinal and colonic neoplasms in high yield in inbred rats. He noted that dimethylhydrazine was structurally similar to cycasin, and hypothesized that both were metabolized to methylazoxymethanol and ultimately to methyldiazonium, a potent alkylating agent.14 Because of their chemical stability, high yield of colon cancers, and short latency period, 1,2-dimethylhydrazine and the related compound azoxymethane have become the most widely used colonic carcinogens. The alkylnitrosamide group, which includes various substituted nitrosoureas and nitrosoguanidines, was initally noted to produce tumors of the upper gastrointestinal tract after oral administration.‘” In 1971, Narisawa et al.“’ showed that direct intrarectal instillation of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in rats caused cancer and adenomatous polyps in the distal

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colon, When injected subcutaneously MNNG produces sarcomas at the site of injection. Ii These results suggest that the carcinogen does not require activation in the liver or by gut bacteria. The ability of aflatoxin, a metabolite of the mold Aspergillus flaws, to produce hepatomas in experimental animals is well known.lH Small amounts of aflatoxin B, added to drinking water also produce benign and malignant colon tumors in a small percentage of vitamin A-deficient rats.‘” This observation is of particular interest in view of the widespread contamination of human foods with these compounds’)” and the report of colon cancer in two laboratory workers involved in the purification of aflatoxin.” Several substituted carbamate? and crude extracts of bracken or fiddlehead fern’:‘, 24also produce small intestinal and colon cancers when fed to experimental animals. However, the tumor yield with these compounds is very low and as a result they have not been extensively studied. Dimethylhydrazine-induced

Colon

Cancer

Because dimethylhydrazine and its metabolites, azoxymethane and methylazoxymethanol, have been the most extensively studied colon carcinogens, this review will focus primarily on studies using these compounds. Rats, mice, and other rodents are excellent test animals because they rarely develop colorectal cancers spontaneously.‘” Dimethylhydrazine or azoxymethane injected weekly at a dosage of 10 to 20 mg per kg of body weight produce colonic adenomas and adenocarcinomas in rats,‘:‘“7 mice,““. 2H and hamsters.:%” At this dosage nearly 100% of animals eventually develop one or more colon tumors, and losses attributable to acute toxicity are negligible. In addition to colon tumors, a significant percentage of rats but not mice develop adenocarcinomas of the duodenum and proximal jejunum and squamous carcinomas of the external ear. A few animals also develop hepatic angiomas and renal cysts. The latency period, defined as the time interval between the first injection of the carcinogen and the appearance of tumors, is approximately 6 months with the above dosage. Reduction of the dose to 7 mg per kg per week lengthens the latency period to approximately 1 year and decreases the number of extracolonic tumors. I4 These compounds are so potent that even a single injection of dimethylhydrazine”’ or azoxymetham? produces colon cancers in rats after a latency period of 15 to 20 months. Another interesting feature of these carcinogens is that the organ distribution of tumors is dependent in part upon the route of administration. For example, addition of dimethylhydrazine to drinking water at a dose of 3 mg per kg per day causes hemangioendotheliomas of the liver rather than tumors.” However, the same total dose given once weekly by gavage instead of daily produces colon cancers in the majority of rats. The age of the animal at the time of injection also influences distribution of tumors. DruckreyU observed that single injections of azoxymethane to l-day-old rats resulted in relatively more neuroblastomas and fewer colon tumors compared to a single injection at 60 days of age. Oral administration of

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cycasin’:’ to pregnant rats produced brain and intestinal tumors in the offspring; however, subcutaneous injection of dimethylhydrazine to pregnant rats was not carcinogenic to the offspring.“” The explanation for these somewhat contradictory results is not obvious. Female rodents are slightly less susceptible than males to the carcinogenic action of dimethylhydrazine:‘“, x and MNNG.‘“’ The clinical and pathological features of dimethylhydrazine-induced colonic neoplasms are very similar to those observed in humans. After 3 to 4 months of treatment the animals develop anorexia, weight loss, and bloody stools. As the colon cancers enlarge they may prolapse through the anus or cause bowel obstruction or intussusception. Metastases to the omentum, lymph nodes, and lung are observed in 20 to 40% of animals, but liver metastases are infrequent. The location of tum.ors in the rat colon is related to the dose of carcinogen. At high doses of azoxymethane (15 mg per kg per week) neoplasms occur predominantly in the left colon, whereas at a lower dose (7 mg per kg per week) the right colon is more involved.“’ This observation may be relevant to human colon cancer which is more frequent in the left colon in high incidence areas and in the right colon in low incidence areas3”, :Sg

Metabolism of Dimethylhydrazine Compounds

and Related

Cycasin, dimethylhydrazine, and azoxymethane are procarcinogens; that is, they require metabolic activation within the host to an active carcinogen. The metabolic activation of dimethylhydrazine’4s 40,4’as shown in figure 2 first involves its oxidation to azomethane, a gas at body temperature which appears in the expired air of dimethylhydrazine-treated rats. A second oxidation converts azomethane to azoxymethane which is then Nhydroxylated to methylazoxymethanol. These metabolic steps probably occur in the liver and possibly in other tissues. Methylazoxymethanol is chemically unstable at body temperature and decomposes spontaneously in vitro to formaldehyde, water, and nitrogen.‘” During this decomposition, the alkylating agent methyldiazonium is formed, which generates a reactive carbonium ion capable of methylating DNA, RNA, or protein.4z Methylazoxymethanol may also undergo enzymatic metabolism in vivo. Grab and Zedek4:’ have recently presented evidence that methylazoxymethanol is converted to methymzoxyformaldehyde by the enzyme alcohol dehydrogenase. Interestingly, this enzyme was present in high concentration in rat liver and colon, which are target organs for this carcinogen, but in very low concentration in jejunum and ileum which are resistant to methylazoxymethanol. Cycasin, the original member of this group, is the pglucoside of methylazoxymethanol (fig. 2). Laqueur and his colleagues showed that cycasin produced colon cancers in rats after oral but not parenteral administration.44 After parenteral injection most of the cycasin was excreted unchanged in the urine, whereas after oral administration only 35% or less was excreted unaltered in the urine, the remainder appearing as

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metabolites in the stool and urine. Furthermore, cycasin was not carcinogenic in germ-free rats when given orally, whereas methylazoxymethanol caused tumors in germ-free or conventional animals when given by any route.‘” These observations indicate that intestinal bacteria hydrolyze the glucose from cycasin and release methylazoxymethanol which decomposes to form the alkylating agent methyldiazonium (fig. 2). The carcinogenicity of cycad flour for humans is unknown. Accurate statistics are not available regarding the incidence of cancer in native Guamanians who consume cycad flour. However, it is unlikely that this flour contains a significant amount of cycasin because the latter is water soluble and is removed during processing of the cycad nuts which are soaked in water for several days before milling.4” The tissues distribution and excretion of dimethylhydrazine have been studied in some detail in an effort to understand the high yield of colon tumors with this compound. Radioautographic studies after injection of [ ‘“Cldimethylhydrazine showed a high uptake of radioactivity in the hepatocytes and endothelial cells of the liver 1 hr after injection and in the epithelial cells of the colon 3 hr after injection.” Pozharisski et al.-‘Hobserved significant alkylation of epithelial cell macromolecules along the entire length of the small and large intestine after injection of [“Hldimethylhydrazine. However, extensive alkylation was also observed in the liver and kidney, organs which rarely develop tumors. Thus it appears that tissue distribution of dimethylhydrazine or its metabolites does not by itself explain the high incidence of colon tumors. The major excretory pathways of dimethylhydrazine and its metabolites are lung and urine, which together account for 40 to 50% of the injected dose.4g The major metabolites in expired air are carbon dioxide and azomethane; urine contains unaltered dimethylhydrazine, azoxymethane, azomethane, and methylazoxymethanol.4U Several investigators”“, x have suggested that dimethylhydrazine is converted to azoxymethane and azoxymethanol in the liver which are then conjugated with glucuronic acid and secreted in bile. The glucuronides could be hydrolyzed by bacterial P-glucuronidase in the colon and finally metabolized to the active carcinogen. Although this pathway may occur, it is not an absolute requirement for subsequent development of colon cancers. For example, Wittig et al.“’ administered dimethylhydrazine to rats after first removing a segment of the distal colon from the fecal stream by performing a diverting colostomy. Colon cancers developed in the defunctionalized segment, although at a slightly reduced incidence compared to nonoperated rats. Zedek et al.“’ demonstrated inhibition of DNA synthesis in the duodenum and colon of rats with a total biliary fistula after injection of methylazoxymethanol. These studies indicate that dimethylhydrazine and its metabolites can be transported to colonic epithelial cells via the blood stream, and do not require biliary excretion to produce tumors. The yield of colon cancers can be modified by compounds which alter the metabolism of dimethylhydrazine. For example, addition of the antioxidant disulfi-

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ram to the diet of dimethylhydrazine-treated mice completely prevents colon cancers.“:1 Fiala et al. demonstrated that disulfirar@ and related compounds4” block the oxidation of azomethane, thereby decreasing the concentration of the active carcinogen. The addition of selenium to the diet also inhibits both dimethylhydrazine and methylazoxymethanol-induced colon cancers by an unknown mechanism.“5 This observation is of interest in view of the suggestion from several epidemiological studies that human cancers are more common in areas of selenium deficiency.5’i. J7 Minor structural modifications of dimethylhydrazine greatly alter its organ specificity. For example, diethylhydrazine, azoethane, and azoxyethane in which an ethyl group is substituted for a methyl group do not produce colon cancers but rather tumors of the brain, spleen, thymus, mammary gland, and liver.‘lxs X’Methylbutylhydrazine and methylazoxybutane produce skin and central nervous system tumors in high yield and colon tumors in much lower yield than the parent compound dimethylhydrazine.‘” These findings led Druckrey’” to speculate that the methyl groups of dimethylhydrazine are important for the high yield of colon tumors. Tissue distribution of the carcinogen may be an important factor in the shift in tumor yield in various organs after alkyl substitution. Thus PozharisskiJX was unable to detect significant alkylation of intestinal epithelial cell macromolecules (DNA, RNA, protein) after injection of diethylhydrazine. However, this compound caused marked alkylation of macromolecules in the spleen, thymus, and brain, organs in which tumors eventually developed. Toth”” has raised the intriguing possibility that synthetic or naturally occurring hydrazines might be involved in human carcinogenesis. Hydrazines as a group are extremely potent carcinogens in experimental animals, and many of them occur in our environment as industrial or food contaminants and as natural constituents of several plants. For example, the substituted hydrazines, l,l-dimethylhydrazine and monomethylhydrazine, are used industrially as components of rocket fuel,“’ whereas 2-hydroxyethylhydrazine is used as a ripener for pineapple and other plants.“” Unsubstituted hydrazine occurs naturally in tobacco,‘i:3 and several types of substituted hydrazines occur in wild and cultivated mushrooms consumed by man.“““4 These interesting observations will hopefully stimulate further studies of the potential role of hydrazines in human neoplasia. Mechanism

of Action and Pathogenesis

Chemical carcinogens produce tumors by modifying the genome of the cell, resulting eventually in alterations of the cell phenotype, such as loss of differentiation, invasiveness, or abnormal proliferative behavior.“” The carcinogenic action of dimethylhydrazine and related compounds appears to involve methylation of colonic epithelial cell DNA. Within hours of injection of dimethylhydrazine, nucleic acids methylated in various positions can be identified in the mouse and rat colonic epithelial cells.““, Ii’ However, methylation of colonic

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DNA is by itself not a sufficient explanation for the high yield of colon cancers with dimethylhydrazine because Pozharisski et al.4x observed significant alkylation in the liver and kidney as well as in the small and large intestine 6 hr after a single dose of dimethylhydrazine. Recent studies with carcinogenic nitrosamines and nitrosamides have suggested that the formation and persistance of O”-alkylguanine (guanine alkylated on the 6-oxygen position) are closely correlated with the eventual formation of tumors in various tissues.“x, ‘i!’ This structural alteration may allow guanine to pair with thymine rather than its normal partner cytosine, thus producing a genetic mutation in subsequent replications. Rogers and Pegg’” observed the formation of substantial amounts of O”-methylguanine in rat colon after a single injection of dimethylhydrazine. However, the liver and kidney, which are not target organs at this dosage, contained even greater amounts of O”methylguanine. It is clear from these studies that factors other than the extent or type of alkylation of DNA must explain the high yield of colon tumors obtained with these compounds. The relatively rapid cell turnover in the colon compared to the liver might also contribute to the high yield of colon tumors. Partial hepatectomy, which greatly increases cell turnover in the remaining liver, is known to enhance the formation of liver tumors after a single dose of dimethylnitrosamine” or methylnitrosourea.” However, cell turnover is also quite rapid in the stomach and distal small intestine, but tumors do not occur in these organs after administration of dimethylhydrazine. Delayed or incomplete repair of damaged DNA in the colon compared to other organs has been suggested as a possible explanation for the high yield of colon tumors with this agent. Kanagalingam and Balis’:’ observed delayed repair of colonic DNA compared to small intestinal DNA after a single dose of dimethylhydrazine. These unrepaired DNA breaks in colonic epithelial cells would presumably enhance tumor formation in the colon. Whatever the mechanisms involved, the colon of rats, like that of man, is inherently more susceptible to carcinogens than the small intestine despite rapid cell turnover in both. This was well illustrated by the interesting experiments of Gennaro et al.‘” in which a segment of the small intestine was transposed to the left colon and a segment of the left colon was transposed to the midsmall bowel of rats. Subsequent treatment of these rats with azoxymethane resulted in a high incidence of cancer in the segment of transposed large intestine, but no tumors occurred in the portion of the small bowel transposed to the colon. These studies indicate that the colonic epithelium is much more susceptible to chemical carcinogenesis than the small intestine and that this susceptibility is probably not related to differences in cell turnover or bacterial flora. The acute and chronic effects of dimethylhydrazine and related compounds on DNA synthesis and cellular proliferation have been studied using biochemical, morphological, and radioautographic techniques. In general, these carcinogens have a biphasic effect on the

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colonic epithelial cell, characterized by an initial acute depression of DNA synthesis lasting several days followed by a prolonged period of increased cellular proliferation. LGhrs et al.‘: showed that a single subcutaneous dose of dimethylhydrazine caused pyknosis and vacuolization of the nuclei of small and large intestinal crypt cells. These acute toxic effects were accompanied by a transient depression of cellular replication which returned to normal 72 to 96 hr after administration of the drug. Similar findings were reported by Zedek et al. 32 who demonstrated that injection of methylazoxymethanol acetate caused a significant depression of DNA synthesis in the colon but not small intestine lasting 72 hr. Intrarectal instillation of dimethylhydrazine caused a profound decrease of the labeling index in the distal colon which was detectable as early as 1 hr after administration of the carcinogen. iti After this acute, toxic depression of DNA synthesis there is an increase in mitotic activity and a widening of the proliferative zone which persists throughout the latent period. These alterations of cellular proliferation are generalized in the colon and occur in epithelium which is morphologically normal. Chan et al.‘” observed a significant increase in the labeling index of the distal colon 24 hr after the intrarectal instillation of a single dose of dimethylhydrazine in mice which persisted for 2 weeks. Similarly, Thurnherr et al.‘x described a widening of the proliferative zone in the colonic epithelium of mice 45 days after initiation of weekly dimethylhydrazine injections. At 97 days there was mitotic activity in morphologically normal surface epithelium and a doubling of the percentage of dividing cells in the colonic crypt. Interestingly, cellular proliferation was normal in the jejunum, an organ not susceptible to tumor formation. An increase in the labeling index does not occur in the dimethylhydrazine-treated rat; however, a widening in the proliferative zone is observed.:’ Similar abnormalities of cellular proliferation have been described in the morphologically normal colonic mucosa of patients with familial colonic polyposis.ix The significance of these changes in cell turnover, particularly their specificity for the preneoplastic state, is presently unknown. Tutton and Barkla have performed detailed studies of cell kinetic parameters and the regulation of cell division in normal and noeplastic rat colonic epithelial cells.7+x:3These investigators initially observed that the biogenic amines, noradrenaline, serotonin, and histamine, accelerated crypt cell division in rat jejunum.% Injection of reserpine, which depletes tissue stores of these compounds, caused a marked depression of cell turnover in both normal colon and colon cancers.80 Certain agents produced a differential effect on cell proliferation in normal colon versus colon cancers. For example, the monoamine oxidase inhibitor nialamide had no effect on cellular proliferation in normal colon but produced increased cell division in tumors.80 Similarly, the tr-adrenergic blocker phentolamine decreased cell proliferation in normal colon but was without effect on tumors., whereas epinephrine inhibited and propran0101 accelerated cell proliferation in tumors but had no

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effect in normal tissue.81 Differential cytotoxicity was observed with 5,6_dihydroxytryptamine, a synthetic analogue of the naturally occurring amine 5-hydroxytryptamine.#” Injection of this compound into rats bearing dimethylhydrazine-induced colon cancers caused widespread necrosis of the tumors but had no significant effect on normal tissues.x” These studies indicate that the regulation of cell proliferation differs greatly in normal versus neoplastic colonic epithelial cells. It may be possible to exploit these differences in designing chemotherapy programs directed against colon cancers. For example, a drug which inhibited cell division in normal colonic epithelium but not in tumor cells would enhance the effectiveness of antineoplastic drugs which are active in dividing cells. Several biochemical alterations occur during the latent period between the administration of the carcinogen and the appearance of tumors which may be relevant to the pathophysiology of the premalignant state. Boffa et al.“” demonstrated a striking increase of several nuclear nonhistone proteins in the colonic epithelial cells but not hepatocytes of dimethylhydrazine-treated rats. The synthesis of these proteins increased after 7 weeks of dimethylhydrazine treatment, well before the appearance of tumors. Alterations in mucus synthesis have also been described in normal-appearing colonic epithelial cells during carcinogenesis. In normal rat colonic epithelial cells there is a predominance of sulfomucins, whereas in the dysplastic colonic epithelium of dimethylhydrazine-treated rats the mucin is rich in sialic acid.x” Similar changes in epithelial mucins have been described in the transitional epithelium surrounding human colon cancers.8ti. xi It is possible that these changes in nucleoprotein and mucin synthesis are simply a reflection of the increased cellular turnover which occurs after dimethyl hydrazine injection. Pathology

of Experimental

Colon Cancers

The morphological evolution of colonic neoplasms has been studied in rats,gcT27mice,‘8T 2yand hamsters”O killed at various times after starting weekly dimethylhydrazine injections. The earliest morphological alteration is the appearance of discrete areas of protuberant surface epithelium which is detectable by scanning electson microscopy.xx, x9 These elevations of the surface epithelium are apparently secondary to an elongation of the crypt and an increase in cell proliferation. The light microscopic changes occurring in the colons of rodents injected weekly with 20 mg per kg of dimethylhydrazine can be summarized as follows. Between 5 and 12 weeks of treatment a decrease in goblet cells and hyperplasia of gland units occurs. Areas of focal atypia appear between 10 and 15 weeks, and severe atypia or carcinomas in situ appear at 14 to 16 weeks. Microscopic adenocarcinomas develop between 12 and 18 weeks, and by 18 to 24 weeks visible adenocarcinomas are present in the majority of animals. Benign adenomatous polyps also develop in the majority of animals and multiple benign and malignant neoplasms in various stages of evolution occur in the same animal. Unequivocal transition of a benign, noninvasive adenoma to an invasive

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carcinoma has not been documented in this animal model. Carcinomas are locally invasive at the earliest stages, whereas adenomas grow to large size without developing features of malignancy.” In contrast, Morso+’ has suggested that most human colon cancers do not arise de novo but result from malignant degeneration of previously benign adenomas. The histopathological features of dimethylhydrazineinduced colonic neoplasms in rats and mice”‘, y2 have been carefully described. Mutinous adenocarcinomas occur primarily as sessile or plaque-like lesions in the proximal half of the colon adjacent to lymphoid follicles. These tumors consist primarily of mucus-laden signet ring cells which invade the serosa and metastasize to regional lymph nodes and peritoneum. In contrast, polypoid adenomas and nonmucinous adenocarcinomas occur more frequently in the distal half of the colon. Polypoid lesions are classified as either polypoid adenocarcinomas or polypoid adenomas containing areas of extreme atypia or carcinoma in situ but without invasion of the stalk. Interestingly, hamsters treated with dimethylhydrazine develop only invasive carcinomas without adenomas.30 A high proportion of ratsa but not miceZx, 2g treated with dimethylhydrazine also develop well differentiated adenocarcinomas in the duodenum and to a lesser extent in the jejunum. The majority of these tumors arise within several centimeters of the entrance of the bile duct, suggesting that an activated carcinogen is secreted in bile. Electron microscopy of experimental colon cancers reveals great variability in size and shape of tumor cells compared to the normal columnar epithelial cell of the colon. Tumor cell microvilli are sparse, blunt or clubshaped, and arranged asymmetrically, and surface glycoproteins, as demonstrated by ruthenium red staining, are greatly reduced compared to normal.Xa, x9 Mucus contentH”,“” is likewise markedly reduced in colon cancers compared to the normal epithelium. LaMont et al.!‘:’previously reported that rat colon cancer cells have a significant decrease in activity of the glycosyltransferase enzymes involved in the synthesis of the carbohydrate portion of colonic mucin. Similar reductions in glycoprotein synthesis have also been described in human colon cancers”“, 95and presumably reflect the dedifferentiated state of the colon cancer cell. We have recently demonstrated a sharp increase in glycoprotein synthesis+‘” and in the specific activity of galactosyltransferasey7 in rat colonic mucosa during late fetal development. These studies indicate that the undifferentiated fetal and neoplastic cells produce low levels of cellular glycoproteins compared to the fully differentiated adult epithelial cell. Tumor

Immunity

The availability of a reproducible colon cancer model has provided an excellent opportunity to study the relationships between immune factors and tumor growth. The majority of studies to date indicate that the antigenic properties of colon cancers, and the hostimmune defenses directed against these tumors, are strikingly similar in animals and humans. Garmaise et

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al.“x reported a tumor-associated antigen in rat colon cancers which was also detectable in the sera of tumorbearing rats and fetal rat tissues, but not in normal rat colon. Abeyounis and Milgromss also demonstrated an antigen in dimethylhydrazine-induced rat colon cancers which was also present in high concentration in fetal intestine, but not in other rat tumors. This antigen was detected in normal colonic epithelium but at a concentration 250-fold less than in the colon cancers. Rat colon antigens were further characterized by Martin et al.““’ who described two glycoprotein antigens in colon cancers also present in trace amounts in goblet cells of the normal colonic mucosa. The tumor-associated antigen was localized by immunofluorescence to the apical membrane of the tumor cell, and in the mucus in the lumen of the glands. These characteristics of rat colon cancer antigens are quite similar to human carcinoembryonic antigen, an antigenic glycoprotein found in colon cancer surface membranes and fetal colon, and in much smaller quantities in normal human colon.““-““’ However, immunological cross-reactivity between rat colon cancer antigens and human carcinoembryonic antigens has not been demonstrated. The cell-mediated immune responses of rats to chemically-induced colon cancers have been carefully studied by Steele and Sji@ren’04-‘ox and resemble in most respects the immune responses of human patients to their colon cancers. Lymphocytes from rats bearing colon cancers induced by either dimethylhydrazine, MNNG, or dimethylaminobiphenyl were cytotoxic against colon cancer target cells induced by all three agents but not against normal rat kidney cells or other types of rat tumors.“” Peripheral lymphocytes from tumor-bearing rats were also cytotoxic to fetal colon cells, but not other fetal tissues.““’ This lymphocytotoxicity could be blocked in vitro by a factor in the sera of tumor-bearing rats.““’ By using serial air-contrast barium enemas of dimethylhydrazine-treated rats, it was possible to demonstrate blocking activity in the sera of rats before a visible tumor had formed, and to show its disappearance after successful tumor excision. Similar studies in humans indicate that circulating lymphocytes from most colon cancer patients are cytotoxic to human colon cancer cells from the same patient or different patients, but not to other tumor cells or normal colon cells. “‘x I”’ Serum from patients with colon cancers”’ and membrane extracts of colon cancers”2 blocked the lymphocytotoxicity. The relationship of these in vitro phenomena to in vivo tumor rejection in man or animals has not been established. The effects of manipulation of the immune system on subsequent tumorogenesis have been the subject of several investigations. Steele and Sj6gren’0x demonstrated that immunization of rats with colonic cancer cells elicited a protective immune response. Rats were first injected in the leg with a suspension of viable tumor cells obtained from an MNNG-induced colon cancer cell line. After a visible tumor developed the leg was amputated, and the same animals were then immunized with irradiated tumor cells from the same cell line. Rats immunized in this fashion were able to reject

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8% of normals contain both high levels of fecal bile acids as well as clostridia species capable of metabolizing the steroid nucleus.‘“Z Although these studies suggest a causal role of dietary fat in human colon cancer, further experimental and epidemiological studies are required to confirm these data and establish the validity of these hypotheses. The minor fecal bile acids in man have not been studies as regards their carcinogenicity in experimental test systems. Wide individual variation of the mean daily excretion of fecal sterols occurs in humans resulting in significant overlap between patients with cancer and normal controls.‘2” The diet of urbanized Western countries differs in many ways other than fat intake from the diet in underdeveloped countries. Our diet contains hundreds of additives, preservatives, fertilizers, pesticides, and organic contaminants from soil and water which are potential colonic carcinogens. A positive correlation between fat intake and colon cancer has also been documented in animal studies. Nigro et al.lZ7 demonstrated that azoxymethane-treated rats fed a diet enriched with 35% beef fat developed twice as many small and large bowel tumors as rats fed regular chow. A greater percentage of animals on the Role of Dietary Fat, Bile Acids, and Bacteria in beef fat diet had metastases to the peritoneum than the Experimental Colon Cancer rats on the regular diet. The tumor-enhancing effect of fat was not limited to animal fat. Thus enrichment of The experimental colon cancer model has been widely the diet with 20% corn oil was just as effective as 20% used to test various hypotheses concerning the complex interrelationship of dietary fat, bile acids, and bacteria beef fat in increasing the yield of colonic neoplasms with dimethylhydrazine.“X The effect of dietary fat in in the etiology of human colon cancer. A positive these studies was not related to nonspecific nutritional correlation between the amount of dietary fat and the effects because body weight was comparable in the incidence of human colon cancer was first noted by Wynder et al.” in 1969. This was more convincingly treated and control groups. Furthermore, the mechadocumented in the studies of various mainland and nism did not appear to involve an alteration of hepatic migrant Japanese populations, which showed a parallel or bacterial metabolism of the carcinogen because a high fat diet also enhanced colon tumor production with increase in colon cancer and animal fat intake after migration from Japan to Hawaii”” and the continental methylnitrosourea, a direct alkylating agent administered intrarectally.“” Reddy et al.‘:“’ suggested that the United States.“’ Hill”x, ‘I!’suggested that the increased animal fat consumption in urbanized Western society increase in colon tumors observed with high dietary fat was attributable to a 2-fold increase in bile salts and caused increased biliary excretion of bile acids and cholestero.1, which are converted by colonic bacteria to sterols in the stools of rats fed 20% corn oil compared to 5% corn oil. secondary bile acids and fecal sterols such as coprostanDietary factors might also alter the bacterial flora of one and coprostanol. These compounds might then be the gut and thereby influence the intraluminal metabfurther metabolized by the colonic flora to steroid carolism of fecal sterols or exogenous carcinogens. For cinogens such as phenanthrenes. Several laboratory example, rats fed a diet enriched with ground beef had and epidemiological observations have been marshalled to support this hypothesis. Deoxycholic acid can be a marked increase in their stools of the bacterial enzymes, nitroreductase, azoreductase, and p-glucuronichemically converted in vitro to 20-methylcholanthrene, a potent carcinogen.“” In 1941, Druckrey et a1.12’ dase, compared to grain-fed rats.‘“‘~ lx2It was speculated that these bacterial enzymes could synthesize endogeshowed th.at the noncarcinogenic bile salt dehydronornous carcinogens in the colon by reducing nitro- and cholene was converted to a weak carcinogen by incubaazocompounds to aromatic amines. Intestinal bacteria tion with bacteria cultured from human stool. It should may also be involved in the metabolism of colonic be noted, however, that a colonic carcinogen has not carcinogens because the tumor yield with dimethylhybeen identified in human stools. Individuals from geodrazine is decreased in germ-free rats, whereas that of graphic areas with a high incidence of colon cancer azoxymethane is increased.‘:‘“, I:$4Careful studies of the excrete more bile acids and neutral sterols in their metabolism of these compounds in conventional and stools than individuals from low incidence areas.‘= germ-free animals are obviously required. Certain bacterial species in human stool can convert The possibility that bile salts might be involved in bile acids into polycyclic aromatic compounds which are potential colonic carcinogens.‘“:‘* I24 Furthermore, the the etiology of colon cancer is based on older studies stools of 75% of patients with colon cancers compared to demonstrating that certain bile salts were carcinogenic a subsequent challenge of the same type of colon cancer cells, whereas rats immunized with other tumor cells were not protected. This study indicates that rat colon cancer antigens are capable of eliciting a specific immune response which is protective against subsequent tumor challenge. However, there is no evidence to date that manipulation of the immune system protects against the development of cancer or is beneficial in an animal that already has cancer. Kroes et al.“:{ studied the effect of antilymphocyte globulin on azoxymethaneinduced colon cancers in rats. Although allogeneic skin grafts survived longer in the rats treated with antilymphocyte globulin, there was no change in the incidence or time of appearance of colon cancers. Bacillus Calmette-G&r-in (BCG), a nonspecific stimulator of the immune system, is an effective antitumor agent in several animals and human cancers.“1 However, administration of BCG to rats previously given a tumorproducing dose of dimethylhydrazine had no effect on the subsequent rate of development, size, or metastatic behavior of colon cancers.“” Intralesional injection of BCG directly into fully developed rectal cancers was likewise without benefit.

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cant effect on the yield of colon tumors. Similarly, when injected into rodents. Injection of deoxycholate, apocholic, or bisnor-5-cholenic acid (of questionable pu- increasing the intestinal transit time of rats with carity) into rats caused sarcomas at the injection site in a thartic doses of magnesium sulfate likewise had no small percentage of animals. ‘9rr’:(i The possibility that effect on colon cancer yield with dimethylaminobiphenyl, a carcinogen which exerts its action within the fecal bile acids might act as intraluminal carcinogens colonic lumen. ‘,‘li or cocarcinogens has been tested directly in the experimental colon cancer model. Narisawa et al.‘3x produced Genetic Factors rectal and colon cancers in rats with a single intrarectal instillation of MNNG, a topical carcinogen. The aniIncreased genetic susceptibility to colon cancer occurs mals were then given a daily intrarectal instillation of in patients with familial colonic polyposis’“‘, ‘-1Xand in 1 mg of either lithocholic or taurodeoxycholic acid in certain families without preexisting polyposis.‘-“‘, ‘x’The peanut oil for 11 months. Rats given the carcinogen plus relative susceptibility or resistance of various strains of bile acids developed approximately 4 times as many mice to dimethylhydrazine carcinogenesis is also genetadenomatous polyps but the same number of cancers as ically transmitted. Evans et al.15’reported that 100% of rats given the carcinogen alone. Instillation of the bile inbred ICR/Ha strain of mice were susceptible to diacids alone did not produce tumors. Cholic, deoxycholic, methylhydrazine carcinogenesis, whereas the DBA/2 and chenodeoxycholic acid also increased the yield of and C57BL/Ha strains were completely resistant to the carcinomas and adenomas in germ-free rats treated same dosage of carcinogen. The susceptible ICR and with MNNG and increased the yield of adenomas in resistant C57BL strains were then cross-bred and the conventional rats.13”, I40Further evidence for the tumorhydrids treated with dimethylhydrazine.Z”5 The yield of promoting effect of bile salts was provided by Chomchai tumors in the hybrids indicated that susceptibility to et a1.14’who surgically implanted the common bile duct the carcinogenic effect of dimethylhydrazine was inherof rats into the midportion of the small intestine, then ited as an autosomal dominant trait, which is also the induced colon tumors with azoxymethane. Animals mode of genetic transmission in human familial colonic with surgically transposed bile ducts developed 2 to 3 polyposis. The mechanism of resistance in these mice times as many colon tumors as nonoperated animals. may be related to absence of an enzyme required for the This effect was attributed to a doubling of fecal bile salt metabolic activation of dimethylhydrazine, or a geneticoncentration in the operated group. The mechanism of cally transmitted resistance of the colonic mucosa to the the tumor-enhancing properties of bile acids in these carcinogen. studies is not known. It is possible that instillation of Nonspecific Injury bile acids into the rectum increases cell turnover, which is known to enhance tumor yield in other experimental The classic experiments of Rous”‘” documented the models.“” It is unlikely that the bile acids themselves promoting effect of nonspecific injury on chemical carwere converted to a carcinogen because rectal instillacinogenesis. A mechanical insult, such as punching a tion of bile salts alone for 1 year did not cause tumors. hole in the ear of a rabbit previously painted with a Furthermore, bacterial metabolism of bile salts was not topical carcinogen, caused many tumors to appear in required because bile salts promoted tumor yield in the region of the healed wound. This effect has been germ-free animals. 13$.14” attributed to the increased cellular proliferation which The effects of cholestyramine on experimental colon accompanies wound healing.““, ‘x’The tumor-promoting cancer appear to be contradictory to the results obtained effect of tissue injury has also been described in animals with bile salts. Coadministration of cholestyramine and treated with dimethylhydrazine. Pozharisski”‘” fashdimethylhydrazine or azoxymethane to conventional or ioned a diverticulum in the cecum of rats using a purse germ-free rats significantly increased the yield of colon string suture. This produced an area of chronic inflamcancers.‘“‘-‘4” These results are unexpected because chomation around the suture and an increase in epithelial lestyramine binds bile salts within the lumen and cell proliferation which lasted for 40 to 50 days after the should prevent their interaction with colonic epithelial operation. The incidence of cecal tumors after dimethcells. Much further work is obviously required to un- ylhydrazine was increased approximately 3-fold in the ravel the complex interrelationships of bile acids, steroperated animals compared to nonoperated controls. ols, bacteria, and colonic carcinogens. Most of the tumors appeared in the area of colonic inflammation around the suture line. A similar clusterDietary Fiber ing of experimental colon cancers around suture lines or colostomy stomas has been reported in rats treated Burkitt”” has implicated a relative deficiency of diedimethylaminobiphenyl,i with dimethylhydrazine,“’ tary fiber as a contributing factor to the high incidence of colon cancer in Western man. A deficiency of fiber MNNG,‘“” and azoxymethane.‘” It is also known that human colon cancers occur with increased frequency in would reduce intestinal transit time, thus allowing colonic suture lines,‘“” at or near the site of ureterosigcarcinogens in the colonic lumen to maintain longer moidostomy,‘“’ and in patients with Crohn’s disease’5x contact with the mucosa. To test this hypothesis Ward or chronic ulcerative colitis.‘“” A possible unifying hyet a1.14”compared the tumor yield of azoxymethanepothesis to explain these observations is that colonic treated rats fed either a low residue diet or a diet cell proliferation in these conditions is increased besupplemented with 20 or 40% cellulose. Cellulose caused a marked increase in fecal weight, but had no significause of chronic injury. Increased proliferation has been

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documented in the colonic mucosa of patients with long standing ulcerative colitis. ‘G”

Summary and Comment Several potent and specific chemical carcinogens produce in laboratory rodents a very accurate animal model of human colon cancer. A considerable body of information is available regarding the mechanism of action of these carcinogens, and the histopathological evolution of colonic neoplasms in animals. The sequence of events leading to colon cancer after administration of dimethylhydrazine can be summarized briefly as follows. Dimethylhydrazine is a procarcinogen which undergoes metabolic activation in the host to the active carcinogen methyldiazonium, an alkylating agent. This activation occurs primarily in the liver, and probably in other tissues as well, but does not require the intestinal flora because germ-free animals also develop colonic tumors when given dimethylhydrazine. The activated carcinogen reaches the colon either by the blood stream or via the fecal stream. The primary effect of the carcinogen in the colonic epithelial cell is methylation of DNA. This is not specific for the colon because methylation of DNA also occurs in the liver and kidney, which are not target organs for dimethylhydrazine. After an initial period of reduced DNA synthesis lasting up to several days, there is a generalized increase in cellular proliferation and a widening of the proliferative zone manifested by the appearance of mitotic figures in the upper third of the crypt and on the surface epithelium. After 2 to 3 months of treatment there is a decrease in goblet cells, hyperplasia of glands, and areas of focal atypia. Microscopic adenocarcinomas and adenomatous polyps appear between 4 and 6 months after start:ing treatment, and eventually produce rectal bleeding or bowel obstruction. The yield of tumors in this animal model is effected by alteration in dietary fat, administration of bile salts and cholestyramine, and nonspecific colonic injury; the mechanism of these interactions remains unexplained. How might the study of colon cancer in rats and mice be useful in our understanding of the pathophysiology of the disease in humans? The animal model provides the opport.unity to study the evolution of colon cancer from the initial damage to epithelial cell DNA in the target tissue to the development of an invasive adenocarcinoma. This experimental approach is obviously not feasible in human subjects. It should be possible to define more carefully the complex alterations of cellular chemistry which characterize the early malignant cell, and in this way devise a biochemical test for the early diagnosis of colon cancer in man. The animal model likewise provides an excellent test system to further study the effects of manipulations of the diet, bacterial flora, or biliary excretions on the incidence of colon cancer. This may prove extremely useful in unraveling the complex role of dietary fat, endogenous sterols, and gut bacteria in tumor formation. Experimental immunotherapy and chemotherapy might also be tested in rodents with colon cancers before clinical trials in humans. Although one must exercise great caution in

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extrapolating the results of animal studies to human disease, these animal models will hopefully expand our knowledge of the fundamental biology of colonic neoplasia and ultimately enhance our ability to diagnose and treat colon cancer in man. REFERENCES 1. Wynder EL, Shigematsu T: Environmental factors of cancer of the colon and rectum. Cancer 20:1520-1561, 1967 2. Wynder EL, Kajitani T, Ishikawa S, et al: Environmental factors of cancer of the colon and rectum. II. Japanese epidemiological data. Cancer 23:1210-1220, 1969 3. Lorenz E, Stewart HL: Intestinal carcinoma and other lesions in mice following oral administration of 1,2,5,6_dibenzanthracene and 20-methylcholanthrene. J Nat1 Cancer Inst 1:17-40. 1941-42 4. Homburger F, Hsuek SS, Kerr CS, et al: Inherited susceptibility of inbred strains of Syrian hamsters to induction of subcutaneous sarcomas and mammary and gastrointestinal carcinomas by subcutaneous and gastric administration of polynuclear hydrocarbons. Cancer Res 32:360-370, 1972 5. Walpole AL, Williams M, Roberts DC: The carcinogenic action of 4-aminodiphenyl and 3:2’-dimethyl-4-aminodiphenyl. Br J Ind Med 9:255-263, 1952 6. Cleveland JC, Litvak SF, Cole JW: Identification of the route of action of the carcinogen 3:2’-dimethyl-4-aminobiphenyl in the induction of intestinal neoplasia. Cancer Res 27:708-714. 1967 7. Navarrete A, Spjut H: Effect of colostomy on experimentally produced neoplasms of the colon of the rat. Lancet 20:14661472, 1967 8. Laqueur GL, Mickelson 0, Whiting MG, et al: Carcinogenic properties of nuts from cycas circidis L. indigenous to Guam. J Nat1 Cancer Inst 31:919-951, 1963 9. Whiting MG: Toxicity of cycads. Econ Bot 17:271-282, 1963 10. Laqueur G: Carcinogenic effects of cycad meal and cycasin, methylazoxymethanol glycoside, in rats and effects of cycasin in germfree rats. Fed Proc 23:1386-1387, 1964 11. Laqueur G: The induction of intestinal neoplasms in rats with the glycoside cycasin and its aglycone. Virchows Arch [Pathol Anat] 340:151-163, 1965. 12. Nagasawa HT, Shirota FN. Matsumoto H: Decomposition of methylazoxymethanol, the aglycone of cycasin in D?O. Nature 236:234-235, 1972 13. Druckrey H, Preussmann R, Matzkies F. et al: Selektive Erzeugung von Darmkrebs bei Ratten durch 1,2-dimethyl-hydrazin. Naturwissenschaften 54:285-286. 1967 14. Druckrey H: Production of colonic carcinomas by 1,2-dialkylhydrazines and azoxyalkanes. In Carcinoma of the Colon and Antecedent Epithelium. Edited by WJ Burdette. Springfield, CC Thomas, 1970, p 267-279 15. Sugimura T, Fujimura S, Baba T: Tumor production in the glandular stomach and alimentary tract of the rat by N-methylN’-nitro-N-nitrosoguanidine. Cancer Res 30:455-465, 1970 16. Narisawa T, Sato T, Hayakawa M, et al: Carcinoma of the colon and rectum of rats by rectal infusion of N-methyl-N’nitro-N-nitrosoguanidine. Gann 62:231-234, 1971 17. Druckrey H, Preussmann R, Ivankovic S, et al: Zur Erzeugung subcutaner Sarkome an Rattan. Z. Krebsforsch 68:87-102, 1966 18. Newberne PM, Butler WH: Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review. Cancer Res 29:236-250, 1969 19. Newberne PM, Rogers A: Rat colon carcinomas associated with aflatoxin and marginal vitamin A. J Nat1 Cancer Inst 50:439448, 1973 food 20. Elmund GK, Brewster TC. Tu AT: Aflatoxins-potent

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23 24

25

26

27

28

29

30

31

32 33

34 35.

36.

37.

38. 39. 40.

41.

42.

43.

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poisons and carcinogenic compounds. J Chem Educ 49:398-399, 1972 Deger GE: Aflatoxin-human colon carcinogenesis? Ann Intern Med 85:204, 1976 Harris PN, Gibson WR, Dillard RD: The oncogenicity of two l,l-diaryl-2-propynyl N-cycloalkylcarbamates. Cancer Res 30:2952-2954, 1970 Evans IA, Mason J: Carcinogenic activity of bracken. Nature 208:913-914, 1965 Hirono I, Shibuya C, Fushimi K, et al: Studies on carcinogenic properties of bracken, Pteridium aquilinum. J Nat1 Cancer Inst 45179-188, 1970 Lingeman CH. Garner FM: Comparative study of intestinal adenocarcinomas of animals and man. J Nat1 Cancer Inst 48:325-346, 1972 Schauer A, Voellnagel T, Wildanger F: Cancerisierung des Rattendarmes durch 1,2-Dimethylhydrazin. Z Gesamte Exp Med 150:87-93, 1969 Ward JM: Morphogenesis of chemically induced neoplasms of the colon and small intestine in rats, Lab Invest 30:505-513. 1974 Thurnherr N, Deschner E, Stonehill E. et al: Induction of adenocarcinomas of the colon in mice by weekly injections of 1,2_dimethylhydrazine. Cancer Res 33:940-945, 1973 Wiebecke B, Krey U, Loehrs U, et al: Morphological and autoradiographical investigations on experimental carcinogenesis and polyp development in the intestinal tract of rats and mice. Virchows Arch [Pathol AnatJ360:179-193, 1973 Winneker RC, Tompkins M, Westenberger P. et al: Morphological studies of chemically induced colon tumors in hamsters. Exp Mol Path01 27:19-34, 1977 Martin MS, Martin F, Justrabo E, et al: Induction de cancers coliques chez le rat par injection unique de 1,2-dimethylhydrazine. Biol Gastroenterol (Paris) 7:37-42, 1974 Druckrey H, Lange A: Carcinogenicity of azoxymethane dependent on age in BD rats. Fed Proc 31:1482-1484, 1972 Spatz M, Laqueur G: Transplacental induction of tumors in Sprague-Dawley rats with crude cycad material. J Nat1 Cancer Inst 38:233-245, 1967 Moon RC, Fricks CM, Schiff LJ: Effect of age and sex on colon carcinogenesis. Proc Am Assoc Cancer Res 17:23-28, 1976 Evans JT, Shows TB, Sproul EE, et al: Genetics of colon carcinogenesis in mice treated with 1,2-dimethylhydrazine. Cancer Res 37:134-136, 1977 Balish E, Shih CN, Croft WA, et al: Effect of age, sex and intestinal flora on the induction of colon tumors in rats. J Nat1 Cancer Inst 58:1103-1106, 1977 Ward JM, Yamamoto RS, Brown CA: Pathology of intestinal neoplasms and other lesions in rats exposed to azoxymethane. J Nat1 Cancer Inst 51:1029-1039, 1973 Haenszel W, Correa P: Cancer of the colon and rectum and adenomatous polyps. Cancer 28:14-24, 1971 DeJong UW, Day NE, Muir CS, et al: The distribution of cancer within the large bowel. Int J Cancer 10:463-477, 1972 Fiala ES: Investigations into the metabolism and mode of action of the colon carcinogen 1,2_dimethylhydrazine. Cancer 36:2407-2412, 1975 Fiala ES, Kulakis C, Bobotas G, et al: Detection and estimation of azomethane in expired air of l-2 dimethylhydrazine treated rats. J Nat1 Cancer Inst 56:1271-1273, 1976 Matsumoto H, Higa HH: Studies on methylazoxymethanol, the aglycone of cycasin: methylation of nucleic acids in vitro. Biochem J 98:20-22c, 1966 Grab DJ, Zedek MS: Organ-specific effects of the carcinogen methylazoxymethanol related to metabolism by nicotinamide adenine dinucleotide-dependent dehydrogenases. Cancer Res 37:4182-4189, 1977

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44. Laquer G, Spatz M: Toxicology of cycasin. Cancer Res 28:22622270, 1968 45. Laquer GL: Carcinogenic effects of cycad meal and cycasin, methylazoxymethanol glycoside. in rats and effects of cycasin in germfree rats. Fed Proc 23:1386-1387. 1964 of 46. Yang MG, Sanger VL, Mickelson 0, et al: Carcmogenicity long-term feeding of cycad husks to rats. Proc Sot Exp Biol Med 127:1171-1175, 1968 studies on the distribu47. Shimizu H, Toth B: Autoradiographic tion of “C-1,2-dimethylhydrazine dihydrochloride and its effects on DNA synthesis in Swiss mice. Gann 66:589-601, 1975 48. Pozharisski KM, Kapustin YM, Likhachev AJ, et al: The mechanism of carcinogenic action of 1,2-dimethylhydrazine (SDMH) in rats. Int J Cancer 15:673-683, 1975 49. Fiala ES, Bobotas G, Kalakis C. et al: Effect of disultiram and related compounds on the metabolism in oiuo of the colon carcinogen 1,2-dimethylhydrazine. Biochem Pharmacol 26:17631768, 1977 50. Weisburger JH: Colon carcinogens: their metabolism and mode of action. Cancer 28:60-70, 1971 51. Wittig VG, Wildner GP, Ziebarth D: Der Einfluss der Ingesta auf die Cancerisierung des Rattendarms durch Dimethylhydrazine. Arch Geschwulstforsch 37:105-115, 1971 52. Zedek MS, Grab DJ, Sternberg SS: Differences in the acute response of the various segments of rat intestine to treatment with the intestinal carcinogen methylazoxymethanol acetate. Cancer Res 37:32-36, 1977 neo53. Wattenberg LW: Inhibition of dimethylhydrazine-induced plasia of the large intestine by disulfiram. J Nat1 Cancer Inst 54:1005-1006, 1975 54. Fiala ES, Bobotas G, Kulakis C, et al: Inhibition of 1,2 dimethylhydrazine metabolism by disulfiram. Xenobiotica 7:59, 1977. 55 Jacobs MM, Burger J, Griffin AC: Inhibitory effects of selenium on 1,2_dimethylhydrazine and methylazoxymethanol acetate induction of colon tumors. Cancer Lett 2:133-138, 1977 56 Jansson B, Malahy MA, Siebert GB: Geographical Distribution of Gastrointestinal Cancer and Breast Cancer and Its Relation to Selenium Deficiency. Proceedings, 3rd International Symposium on Detection and Prevention of Cancer. New York, Marcel Dekker Inc, 1976 57. Shamberger RJ, Willis CE: Selenium distribution and human cancer mortality Clin Lab Sci 2:211-216, 1971 58 Druckrey H, Preussman R, Ivankovic S. et al: Carcinogene wirkung von azoaethan und azoxyaethan and ratten. Z Krebsforsch 67:31-45, 1965 59. Druckrey H, Preussman R, Matzkies R, et al: Carcinogene Wirkung von 1,2-Diaethylhydrazin an Ratten. Naturwissenschaften 53:557-558, 1966 60. Toth B: Synthetic and naturally occurring hydrazines as possible cancer causative agents. Cancer Res 35:3693-3697, 1975 61. The Merck Index. Eighth edition. Rahway NJ, Merck and Co Inc, 1968 62. Gowing DP, Leeper RW: Induction of flowering in pineapple by beta-hydroxyethylhydrazine. Science 122:1267, 1955 63. Liu YY, Schmeltz I, Hoffmann D: Chemical studies on tobacco smoke. Quantitative analysis of hydrazine in tobacco and cigarette smoke. Anal Chem 46:885-889, 1974 64. Levenberg B: Isolation and structure of agaritine, a y-glutamylsubstituted arylhydrazine derivative from Agaricaceae. J Biol Chem 239:2267-2273, 1964 65. Ryser HJP: Chemical carcinogenesis. N Engl J Med 285:721734, 1971 66. Hawks A, Swann PF, Magee PN: Probable methylation of nucleic acids of mouse colon by 1,2_dimethylhydrazine in ho. Biochem Pharmacol21:432-435, 1971 67. Hawks A, Magee PN: The alkylation of nucleic acids of rat and

December 1975

68. 69

70

71

72

73 74.

PROGRESS

mouse in uiuo by the carcinogen 1,2 dimethylhydrazine. Br J Cancer 30:440-446, 1974 Goth R, Rajewsky M: Persistance of O”-ethylguanine in rat brain DNA. Proc Nat1 Acad Sci USA 71:639-643, 1974 Margison GB, Kleihues P: Chemical carcinogenesis in the nervous system. Preferential accumulation of O”-methylguanine in rat brain DNA during repetitive administration of Nmethyl-N-nitrosourea. Biochem J 148:521-525, 1975 Rogers KJ, Pegg AE: Formation of O”-methylguanine by alkylation of rat liver. colon and kidney DNA following administration of 1.2-dimethylhydrazine. Cancer Res 37:4082-4087, 1977 Craddock VM: Liver carcinomas induced in rats by a single administration of dimethylnitrosamine after partial hepatectomy. J .Natl Cancer Inst 47:889-907, 1971 Craddock VM, Frei JV: Induction of liver cell adenomata in the rat by a single treatment with N-methyl-N-nitrosourea given at various times after partial hepatectomy. Br J Cancer 30:503511, 1974 Kanagalingam K, Balis EM: In uiuo repair of rat intestinal DNA damage by alkylating agents. Cancer 36:2364-2372, 1975 Gennaro AR, Villanueva R, Sukonthaman Y, et al: Chemical carcinogenesis in transposed intestinal segments. Cancer Res 33:536-541, 1973

75. Loehrs 1J. Wiebecke B, Eder M: Morphologische undautoradiographische Untersuchung der Darmschleimhautveranderungen nach einmaliger Injektion von 1,2_Dimethylhydrazine. Z Gesamte Exp Med 151:297-307, 1969 76. Chan PC, Cohen LA, Narisawa T, et al: Early effects of a single intrarectal dose of 1,2-dimethylhydrazine in mice. Cancer Res 36:13-li. 1976 77. Maskens AP: Histogenesis and growth pattern of 1,2-dimethylhydrazine-induced rat colon adenocarcinoma. Cancer Res 36:1585--1592, 1976 78. Deschner E, Lewis CM, Lipkin M: In vitro study of human rectal epithelial cells. 1. Atypical zone of :‘H-thymidine incorporation in mucosa of multiple polyposis. J Clin Invest 42:19221928, 1963 79. Tutton PJ, Barkla DH: Cell proliferation in the descending colon of dimethylhydrazine treated rats and in dimethylhydrazine induced adenocarcinomata. Virchows Arch [Zellpathol] 21:147- 160, 1976 80. Tutton PJ. Barkla DH: A comparison of cell proliferation in normal and neoplastic intestinal epithelia following either biogenic amine depletion or monoamine oxidase inhibition. Virchows Arch [Zellpathol] 21:161-168, 1976 81 Tutton PJ. Barkla DH: The influence of adrenoceptor activity on cell proliferation in colonic crypt epithelium and in colonic adenoc.arcinomata. Virchows Arch [ZellpatholJ 24:139-164, 1977 82 Tutton PJ, Barkla DH: Cytotoxicity of 5,6_dihydroxytryptamine in dimethylhydrazine-induced carcinomas of rat colon. Cancer Res 37:1241-1244, 1977 83. Tutton PJ. Heline RD: The influence of adrenoreceptor activity on crypt cell proliferation in the rat jejunum. Cell Tissue Kinet 7:125-136, 1974 84. Boffa LC, Vidali G. Allfrey VG: Changes in nuclear nonhistone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells. Exp Cell Res 98:396--410, 1976 85, Filipe MI: Mucous secretion in rat colonic mucosa during carcinogenesis induced by dimethylhydrazine. A morphological and hifstochemical study. Br J Cancer 32:60-77. 1975 86. Filipe MI, Cooke KB: Changes in the composition of mucin in the mucosa adjacent to carcinoma of the colon as compared with the normal. J Clin Path 27:315-322. 1974 87. Filipe MI, Branfoot AC: Abnormal patterns of mucus secretion in apparently normal mucosa of large intestine with carcinoma. Cancer 34:282-289, 1974

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88. Barkla DH, Tutton PJ: Surface changes in the descending colon of rats treated with dimethylhydrazine. Cancer Res 37:262-271, 1977 89. Toth B, Malick L: Production of intestinal and other tumors by 1,2_dimethylhydrazine dihydrochloride in mice. II. Scanning electron microscopic and cytochemical study of colonic neoplasms. Br J Exp Path01 57:696-705, 1976 90. Morson BD: Evolution of cancer of the colon and rectum. Cancer 34:845-860, 1974 91. Ward JM, Yamamoto RS, Benjamin T, et al: Experimentally induced cancer of the colon in rats and mice. J Am Vet Med Assoc 164:729-732, 1974 92 Haase P, Corven DM, Knowles JC, et al: Evaluation of dimethylhydrazine induced tumors in mice as a model system for colorectal cancer. Br J Cancer 28:530-543, 1973 93 LaMont JT, Weiser MM, Isselbacher KJ: Cell surface glycosyltransferase activity in normal and neoplastic intestinal epithelium of the rat. Cancer Res 34:3225-3228, 1974 94. Kim YS, Isaacs R, Perdomo J: Alterations of membrane glycopeptides in human colonic adenocarcinoma. Proc Nat1 Acad Sci USA 71:4869-4873, 1974 95 LaMont JT, Isselbacher KJ: Alterations in glycosyltransferase in human colon cancer. J Nat1 Cancer Inst 54:53-56, 1975 96. Rampal P, LaMont JT, Trier JS: Differentiation of glycoprotein synthesis in fetal rat colon. Am J Physiol 235:E207-E212, 1978 97. LaMont JT, Ventola AS: Galactosyltransferase in fetal, neonatal and adult colon: relationship to differentiation. Am J Physiol 235:E213-E217, 1978 98. Garmaise AB, Rogers AE, Saravis C, et al: Immunologic aspects of 1,2-dimethylhydrazine-induced colon tumors in rats. J Nat1 Cancer Inst 54:1231-1235, 1975 99. Abeyounis CJ, Milgrom F: A thermostable antigen characteristic for carcinoma-induced rat intestinal tumors. J Immunol 116:30-34, 1976 100. Martin F, Martin MS, Bordes M, et al: Antigens associated with chemically induced intestinal carcinomas of rats. Int J Cancer 15144-151, 1975 101. Gold P, Freedman SO: Specific carcino-embryonic antigens of the human digestive system. J Exp Med 121:439-462, 1965 102. Gold P, Krupey J, Ansari H: Position of the carcinoembryionic antigen of the human digestive system in ultrastructure of tumor cell surface. J Nat1 Cancer Inst 45:219-225, 1970 103. Rosai J, Tillack TW, Marchesi VT: Membrane antigens of human colonic carcinoma and non-tumoral colonic mucosa: results obtained with a new isolation method. Int J Cancer 10:357-367, 1972 104. Steele G, Sjoegren HO: Cross-reacting tumor-associated antigen(s) among chemically induced rat colon carcinomas. Cancer Res 34:1801-1807, 1974 105. Steele G, Sjoegren HO: Embryonic antigens associated with chemically induced colon carcinomas in rats. Int J Cancer 14:435-444, 1974 106. Steele G, Sjoegren HO, Rosengren JE, et al: Sequential studies of serum blocking activity in rats bearing chemically induced primary bowel tumors. J Nat1 Cancer Inst 54:959-967, 1975 107. Steele G, Sjoegren HO, Price MR: Tumor-associated and embryonic antigens in soluble fractions of a chemically-induced rat colon carcinoma. Int J Cancer 16:33-51. 1975 108. Steele G, Sjoegren HO: Cell surface antigens in a rat colon cancer model: correlation with inhibition of tumor growth. Surgery 82:164-169, 1977 109. Hellstroem I, Hellstroem KE, Shepard TH: Cell-mediated immunity against antigens common to human colonic carcinomas and fetal gut epithelium. Int J Cancer 6:346-351. 1970 110. Hellstroem I, Hellstroem KE, Sjoegren HO, et al: Demonstration of cell-mediated immunity to human neoplasms of various histological types. Int J Cancer 9:1-16, 1971

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111. Hellstroem I, Sjoegren HO, Warner GA, et al: Blocking of cellmediated tumour immunity by sera from patients with growing neoplasms. Int J Cancer 7:225-237, 1971 112. Baldwin RW, Embleton MJ, Price MR: Inhibition of lymphocyte cytotoxicity for human colon carcinoma by treatment with solubilized tumour membrane fractions. Int J Cancer 12:84-92. 1973 113. Kroes R, Berkvens JM. Weisburger JH: Immunosuppression in primary liver and colon tumor induction with N-hydroxy-N-2fluorenylacetamide and azoxymethane. Cancer Res 35:26512656, 1975 114. Bast RC, Zbar B, Borsos T, et al: BCG and cancer. N Engl J Med 290:1413-1420, 14581469, 1974 115. Rogers AE, Gildin J: Effect of BCG on dimethylhydrazine induction of colon tumors in rats. J Nat1 Cancer Inst 55:385391, 1975 116. Haenszel W, Berg JE, Segi M: Large bowel cancer in Hawaiian Japanese. J Nat1 Cancer Inst 51:1765-1799, 1973 117 Haenszel W, Kurihara M: Studies of Japanese migrants. 1. Mortality from cancer and other chronic diseases among Japanese in the United States. J Nat1 Cancer Inst 40:43-68, 1968 118. Hill MJ: Gut bacteria, steroids and cancer of the large bowel. In Some Implications of Steroid Hormone in Cancer. Edited by DC Williams, MH Briggs. London, William Heineman Medical Books Ltd, 1971, p 94-106 119. Hill MJ, Aries VC: Fecal steroid composition and its relationship to cancer of the large bowel. J Path01 Bact 104:129-139, 1971 120. Cook JW, Haslewood GA: Conversion of a bile acid into a hydrocarbon derived from 1,2-benzanthracene. Chem Indust 11:758-759, 1933 121. Druckrey H, Richter R, Bierthaler R: Zur endogenen Entstehung krebserregender Stoffe beim Menschen. Klin Wochenschr 20:781-785, 1941 122. Reddy BS, Wynder EL: Large bowel carcinogenesis: fecal constituents of populations with diverse incidence rates of colon cancer. J Nat1 Cancer Inst 50:1437-1442, 1973 123. Aries VC, Goddard P, Hill MJ: Degradation of steroids by intestinal bacteria. III. 3-oxo-5P-steroid A’-dehydrogenase and 3-oxo-5P-steroid A’-dehydrogenase. Biochim Biophys Acta 248482-488, 1971 124. Goddard P, Hill MJ: Degradation of steroids by intestinal bacteria. IV. Aromatisation of ring A. Biochim Biophys Acta 280:336-342, 1972 125. Hill MJ, Aries VC: Faecal steroid composition and its relationship to cancer of the large bowel. J Path01 Bact 104:129-139, 1971 126. Reddy BS, Wynder EL: Metabolic epidemiology of colon cancer. Fecal bile acids and neutral sterols in colon cancer patients and patients with adenomatous polyps. Cancer 39:2533-2539, 1977 127. Nigro ND, Singh DV, Campbell RL, et al: Effect of dietary beef fat on intestinal tumor formation by azoxymethane in rats. J Nat1 Cancer Inst 54:439-442, 1975 128. Reddy BS, Narisawa T, Vukusich D, et al: Effect of quality and quantity of dietary fat and dimethylhydrazine in colon carcinogenesis in rats. Proc Sot Exp Biol Med 151:237-239, 1976 129. Reddy BS, Watanabe K, Weisburger JH: Effect of high-fat diet on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine, methylazoxymethanol acetate, or methylnitrosourea. Cancer Res 37:4156-4159, 1977 130. Reddy BS, Weisburger JH, Wynder EL: Effects of dietary fat level and dimethylhydrazine on fecal acid and neutral sterol excretion and colon carcinogenesis in rats. J Nat1 Cancer Inst 52:507-511, 1974 131. Goldin BR, Gorbach SL: The relationship between diet and rat fecal bacterial enzymes implicated in colon cancer. J Nat1

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Cancer Inst 57:371-375, 1976 132 Reddy BV, Mangat S, Weisburger JH, et al: Effect of high risk diets for colon carcinogenesis on intestinal mucosal and bacterial p-glucuronidase activity in F 344 rats. Cancer Res 37:35333536, 1977 133 Reddy BS, Narisawa T, Wright P, et al: Colon carcinogenesis with azoxymethane and dimethylhydrazine in germ-free rats. Cancer Res 35:287-290, 1975 134. Reddy BS, Narisawa T, Weisburger JH: Colon carcinogenesis in germ-free rats with intrarectal 1,2_dimethylhydrazine and subcutaneous azoxymethane. Cancer Res 36:2874-2876, 1975 135. Cook JW, Kennaway EL, Kennaway NM: Production of tumors in mice by deoxycholic acid. Nature 145:627, 1940 136. Lacassagne A, Buu-Hoi NP, Zaidela F: Carcinogenic activity of apocholic acid. Nature 190:1007-1008, 1961 137. Lacassagne A, Buu-Hoi NP, Zaidela F: Carcinogenic activity in situ of further steroid compounds. Nature 209:1026-1027, 1966 138. Narasawa T, Magadia NE, Weisburger JH, et al: Promoting effects of bile acids on colon carcinogenesis after intrarectal installation of N-methyl-N’-nitro-N-nitrosaguanidine in rats. J Nat1 Cancer Inst 53:1093-1097, 1974 139. Reddy BS, Narisawa T, Weisburger JH, et al: Promoting effect of sodium deoxycholate on colon adenocarcinomas in germfree rats. J Nat1 Cancer Inst 56:441-442, 1976 140. Reddy BS, Watanabe K, Weisburger JH, et al: Promoting effect of bile acids in colon carcinogenesis in germ-free and conventional F344 rats. Cancer Res 37:3238-3242, 1977 141. Chomchai C, Bhadrachari N, Nigro ND: The effect of bile on the induction of experimental intestinal tumors in rats. Dis Colon Rectum 17:310-312, 1974 N, Chomchai C: A rat model for 142. Nigro ND, Bhadrachari studying colon cancer. Effect of cholestyramine on induced tumors. Dis Colon Rectum 16:438-443, 1973 143. Asano T, Pollard M, Madsen DC: Effects of cholestyramine on 1,2-dimethylhydrazine-induced enteric carcinoma in germfree rats. Proc Sot Exp Biol Med 150:780-785, 1975 144. Burkitt DP: Epidemiology of cancer of the colon and rectum. Cancer 28:3-13, 1971 145. Ward JM, Yamamoto RS, Weisburger JH: Cellulose dietary bulk and azoxymethane-induced intestinal cancer. J Nat1 Cancer Inst 51:713-715, 1973 146. Cleveland JC, Cole JW: Relationship of experimentally induced intestinal tumors to laxative ingestion. Cancer 23:1200-1203, 1969 147. Bockus HL, Tachdjian V, Ferguson LK, et al: Adenomatous polyps of colon and rectum: its relation to carcinoma. Gastroenterology 41:225-232, 1961 148. McKusick VA: Genetic factors in intestinal polyposis. JAMA 182:271-277, 1962 149. Savage D: A family history of uterine and gastrointestinal cancer: Br Med J 2:341-343, 1956 150. Lynch HT, Hoda G, Swartz M, et al: Genetics and colon cancer. Arch Surg 106:669-675, 1973 151. Evans JT, Hauschka TS, Mittelman A: Differential susceptability of four mouse strains to induction of multiple large-bowel neoplasms by 1,2_dimethylhydrazine. J Nat1 Cancer Inst 52:999-1000, 1974 152. Rous P, Kidd JG: Conditional neoplasms and subthreshold neoplastic states: a study of the tar tumors of rabbits. J Exp Med 73:365-390, 1941 153. Pullinger BO: A measure of the stimulating effect of simple injury combined with carcinogenic chemicals on tumour formation in mice. J Path01 Bact 57:477-481, 1945 154. Pozharisski KM: The significance of nonspecific injury for colon carcinogenesis in rats. Cancer Res 353824-3830, 1975 155. Kanazawa K, Yamamoto T, Sato S: Experimental induction of

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1978

PROGRESS

colonic carcinomas in rats. Jap J Exp Med 45439-456, 1975 156. Floyd CE, Corley RG, Cohn I: Local recurrence of carcinoma of the colon and rectum. Am J Surg 109:109-128, 1965 157. Mueller CE, Thornbury JR Adenocarcinoma of the colon complicating ureterosigmoidostomy: a case report and review of the literature. J Urol 109:225-227. 1973

ARTICLE

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158. Weedon DD, Shorter RG, Ilstrup DM, et al: Crohn’s disease and cancer. N Engl J Med 289:1099-1103, 1973 159. Morson BC: Cancer in ulcerative colitis. Gut 7:425-426, 1966 160. Bleiberg H. Mainguet P, Galand P, et al: Cell renewal in the human rectum. In vitro autoradiographic study on active ulcerative colitis. Gastroenterology 58:851-855, 1970