Cancer Letters, 61 (1992) 177 - 183 Elsevier Scientific Publishers Ireland Ltd.
177
Induction of colorectal cancer in rats by ZO-methylcholanthrene R.N. Baral and P. Maity Department of Cell Biology, Chittoranjon (Received (Accepted
National
Cancer
Institute, 37, S.P. Mukherjee
700 026 (India)
Colorectal
Introduction carcinoma
was
induced
Sprague - Dawley rats by 20-methylcholanthrene. Macroscopical studies revealed that tumors, either sessile type or semi-peduncul-
in
Colorectal carcinoma has been induced in mice and rats by different carcinogenic agents, like cycasin, methylazoxymethanol (MAM) (DMH) [8,9], [l&19], 1,2-d imethylhydrazine IV-methyl-N’nitro-IV-nitrosoguanidine (MNNG) [Zl], N-methyl-N-nitrosourea (MNU) [20] etc. and in most of the cases, appearance of the carcinoma were reported within 25 weeks of the treatment [2,8,9,16,18 - 211. 20-Methylcholanthrene (MCA) was considered as a potent cancer causing agent [12,17,26]. Sarcoma in rats produced by MCA was described by Huggins et al. in 1963 [15]. Homburger et al., in their elaborate survey of chemical carcinogenesis found that oral administration of MCA in hamsters causes colon cancer [ 131. Carcinogenic effects of 20-MCA in the mice large intestine were also reported [1,23]. In 1958, Horava et al. described cecoappendiceal carcinoma in rats by local application of the 20-MCA, however, no tumors were found by them in the colons of experimental animals [ 141. In the present investigation, we intend to examine, whether colorectal cancer can be produced in rats by direct application of MCA through the rectum.
the
ated polyp, were generally observed after 32 weeks of the carcinogen treatment. In the distal colon 46.9% tumors appeared, whereas
20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively. Sequential histiopathological studies indicated that hyperplaria of goblet cells was common in early stages, which was reduced thereafter. Carcinogenesis progressed with the appearance of the different grades of dysplasia in colmucosa dysplasia
wifh first incidence of the in rats at the 20th week and
carcinoma at the close of 28th week. of the carcinomas were multifocal in origin and were well differentiated adenocarcinema with primary invasion at the submucosa. In immunohistological studies, this carcinoma was also reactive with monoclonal in situ Most
antibody carcinoma
660, prepared against a co/ore&al associated mucin antigen.
colorectal Keywords: methylcholanthrene; tumors;
carcinogenesis; adenocarcinoma
Materials Correspondence to: P. Maity, Department of Cell Biology, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Calcutta
Calcutta
19 September 1991) 1 October 1991)
Summary
ore&al severe
Road.
Carcinogenesis
700 026, India.
0304.3835/92/$05.00 Printed and Published
G 1992 Elsevier Scientific Publishers in Ireland
and Methods
Seventy male Sprague - Dawley rats, 8 weeks Ireland
Ltd
178
of age, weighing approximately 130 g at the beginning of the experiment, were selected for this study and randomly divided into three groups. A group of 40 rats received MCA dissolved in benzene, another group of 20 rats was treated with benzene alone and 10 rats were maintained as normal control. For colorectal carcinogenesis, each rat received 2 mg MCA per week. Cotton threads soaked with 0.5% MCA solution (1 mg MCA + 200 ~1 benzene) were placed into the lumen of the large intestine through anal orifices of the experimental rats and carcinogen was inserted twice a week. A group of animals received benzene only in the same way as the carcinogen-treated group. At a regular interval of 4 weeks, rats from each group (5 from MCA + benzene; 2 from benzene alone and 1 from normal) were sacrificed for histological study. The experiment was continued up to 32 weeks. Histology The colon and rectum of animals that had been treated with MCA for different periods were opened, cleaned in phosphate buffered saline (PBS), pH 7.2, and examined for the size and distribution of tumors. number, Thereafter, the rectum, distal and proximal colon of control as well as experimental animals were fixed in 95% ethanol [5] and coiled up into rolls [lo]. Tissues were processed by routine histological methods and sections were prepared. Each section was stained with haematoxylin-eosin and also studied immunohistochemically. Histopathological staging Preneoplastic and neoplastic lesions were classified on the basis of histological differentiation according to the method described by M.I. Filipe [lo] and Decaens et al. [5]. lmmunohistochemistry A monoclonal antibody (MAb 660), against a mucin-associated 660 antigen, was used in this experiment. MA\, 660 was received as a gift from Dr. C. Decaens of Mucin Immunochemistry Laboratory, Villejuif, Cedex, France.
Sections were deparaffinised in toluene and endogeneous peroxidase activity was inhibited by incubation of sections with 0.5% H202 in methanol for 20 min. After thorough washing in PBS, tissue sections were incubated overnight at 4OC with MAb 660. Incubated sections, after washing with PBS, were incubated again with sheep antiserum against mouse IgG (H + L) labelled with peroxidase (Diagnostics Pasteur, Marnes La Coquette, France) at a l/100 dilution for 45 min. The sections were washed with PBS and peroxidase activity was revealed using aminoethylcarbazol according to the method of Graham et al. [ll]. Slides were counter stained with haematoxylin before microscopic examination. Statistics The Scheffe’s F-test was used to determine the significance of differences between the number of tumors in the rectum, distal and proximal colon. Results Macroscopy The gross appearance of the luminal surface of the distal colon was morphologically abnormal in the late stages of the carcinogenesis, but very few animals possessed tumorous growth before 32nd week of the treatment. All rats sacrificed after 32nd week showed the presence of tumors within the large intestinal lumen (Table I). Most of the growths (Fig. l), found in the colorectum, were of the sessile type with a smooth surface structure, but a few tumors were semipedunculated polyps and a single cauliflower-like growth was observed in the rectum of one induced animal. Histopathology The histopathologic findings are shown in Table II. Mucosal changes following MCA administration had been classified into hyperplastic foci, different grades of dysplasia and carcinoma. Hyperplasia Epithelial
hyperplasia
was
observed
in
179
Table 1. Distribution, size and number of tumors appeared in the colorectum of five rats treated with MCA for 32 weeks. Sites
Tumor size (mm)
Number of tumors
Rectum
55 5.1-9.9 2 10 55 5.1-9.9 2 10 55 5.1-9.9 2 10
3 4 3 8 9 6 4 7 5
Distal colon
Proximal colon
‘Significantly
different
from rectum,
Total number of tumors (%)
10 (20.4)
23’
(46.9)
16 (32.6)
P < 0.01.
animals treated with MCA for 4- 12 weeks. The colorectal tissues of these animals showed dilated crypts due to marked increase in the secreting goblet cells. mucous A few hyperplastic glands were mildly hyperchromatic in animals that had received MCA for more than 8 weeks, but the nuclei were aligned regularly on the basement membrane.
Fig. 1.
Macroscopic
tumors
appeared
Dysplasia divided into different Dysplasias were groups according to the severity of the disease, i.e. from grade 0 to grade 3. Rats received carcinogen not more than 16 weeks, were included in the group of dysplasia grade 0. In this grade, crypts may be dilated, but a significant reduction in the number of goblet cells were seen. This reduction in the cellular activity of the goblet cells was progressed with the advancement of the disease. Animals after 16 weeks of carcinogenesis showed features of Dysplasia grade 1, like cellular atypia, however, nuclear polarity was unchanged in these animals. In the 24th and 28th week, animals showed irregular tubular structure, with loss of nuclear polarity and appearance of the mitotic figures (Dysplasia grade 2). Animals that received more than 28 weeks treatment possessed dysplastic glands of the third grade. Back-to-back architecture along with severe glandular atypia were seen in most of these animals. Carcinoma
The malignant neoplasms observed in our experiments generally after 32 weeks of carcinogen insertion, were either in situ or in(Fig. 2). These are filtrating carcinomas
at distal (D) and proximal
(P) colon of rat treated
with MCA for 32 weeks.
180
Table II. Time of appearance Weeks of treatment
4 8 12 16 20 24 28 32
Features
of different
carcinogenic
x
carcinogenesis.
Goblet cellular proliferation
Hyperchromatism
Atypism
Loss of polarity
Expression of mitotic figures
+++ +++ +++ ++ ++ f f -
+ + + + +/+ ++ +++ +++ +++
Not observed do do +/+ + ++ +++ +++ +++
Not observed do do + ++ +++ +++ +++
As normal do do ++ ++ +++ +++ +++
and marked
exp:ession
characterized by loss of glandular architecture with different degrees of differentiation. Most of the carcinomas were multifocal in origin and were well-differentiated adenocarcinoma, although a few mucosal foci with moderate or poor differentiation were also demonstrated. Carcinomas often reached a depth of submucosa, but only two incidents were demonstrated where carcinomas invaded to muscular layer. Areas of hyperplasia and dysplasias were
Fig. 2.
during colorectal
of carcinogenesis
+ , + + , + + + = mild, moderate
Eosin,
features
An ulcerated 120.
adenocarcinoma
at the rat rectum
of different
carcinogenic
features.
often adjacent to the carcinoma invasive carcinomas.
in situ and
Controls The changes observed in rats treated with benzene alone were not comparable with the MCA treated groups. The only change that appeared in the mucosa was occasional mild glandular hyperplasia. The pattern of crypts were completely unaltered. The group of ten animals used as normal
after 32 weeks of the MCA treatment.
Haematoxylin
and
181
control showed neither macroscopical growth nor microscopical lesions in the colorectum. lmmunohistochemistry
The study with MAb 660 was performed on the colorectal mucosae of normal and MCA treated rats. Most of the large intestinal carcinomas appeared in our studies were reactive with MAb 660. This antigen was also expressed in dysplastic (Fig. 3)) hyperplastic as well as apparently normal mucosal glands of MCA induced animals. However, MAb 660 reactivity was absent in colorectal mucosae of normal rats. Discussion
To our knowledge, we are the first to report the induction of colorectal carcinoma in Sprague - Dawley rats by direct application of MCA through the rectum. Although Horava et al. tried to develop tumors at colon by the
Fig. 3. A dysplastic gland of distal colon, adjacent to carcinoma. exhibiting 660 antigen, immunoperoxidase. x480.
same carcinogen, but they were not successful in that attempt [14]. Macroscopical studies that revealed the incidence of tumors in 100% of the rats occurred only after 32 weeks of treatment. M.I. Filipe reported macroscopical lesions at the colon after 19 weeks in all the DMH-treated rats [lo]. Narisawa demonstrated the same within 25 weeks during carcinogenesis with MAM [22]. In comparison, the animal system described here, took longer time to produce tumorous growth. This slow progression of the disease may not be due to low dose of the MCA. An animal, undergoing treatment for 32 weeks, received a total dose of 64 mg of MCA and this dose was comparatively higher than the dose described in DMH, MAM, MNNG and MNU induced systems [6,7,20,24,25]. The maximum number of tumors (46.9%) appeared in the distal colon after 32 weeks of MCA instillation, whereas 20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively (Table I). Narisawa et al. also induced many tumors in the distal large bowel of rats by the intrarectal administration of MNNG and MAM [21,22]. This part of the colon was also described as a most sensitive site of occurrence of malignant growth in other experimental models [7,10,25]. Different features of carcinogenesis in relation to duration of treatment are presented in Table II. The earlier change, detected after 4 weeks, was cellular hyperplasia of goblet cells, which was reduced linearly with the progression of the disease. This observation correlates well with the features reported in a similar stepwise investigation carried out by Thurnherr et al. [27] and M.I. Filipe [lo]. Severe dysplasia and in situ carcinoma were observed after 13th week and 15th week respectively during DMH induced carcinogenesis [lo], whereas, in the present study, severe dysplasia was first seen in an animal at the 20th week and in situ carcinoma at the end of the 28th week. The malignant neoplasms produced in our experiments were adenocarcinoma with difof differentiation. Several ferent degrees groups of investigators reported this type of
182
carcinoma in their experimental system during carcinogenesis with a variety of carcinogens [7,10,16,20,22]. In human system also, is the most common adenocarcinoma malignancy of the large gut [3]. MCA-induced carcinoma invaded mainly to the submucosa and this finding was similar to the nature of observed in other experimental invasion models [10,20,22]. The expression of an oncofetal mucin antigen, the 660 antigen, in the preneoplastic as well as neoplastic colorectal mucosae during MCA-induced carcinogenesis was similar to the results described by Decaens et al. [4] and this observation provides an additional support in favour of carcinogenic changes produced by MCA. It may be concluded from our results that a new experimental model of slow progressing colorectal carcinoma has been developed in rats by applying MCA locally and this model may help us in the understanding of the mechanism of carcinogenesis in human beings, where it is a long-term process. Acknowledgements authors thank Dr. J. Roychowdhury, Director, CNCI, Calcutta, for providing all facilities. We wish to express our thanks to Dr. R.N. Chakraborty for his helpful suggestions in histopathological studies. We are also indebted to Dr. C. Decaens for her gift of the monoclonal antibody. The first author gratefully acknowledges the financial assistance given by Indian Council of Medical Research, New Delhi.
4
Decaens, C., Gautier, R., Bara, J., Daher, N.. Pendu, J.L. and Burtin, P. (1988) A new mucin associated oncofetal antigen, a marker of early carcinogenesis in rat colon. Cancer Res., 48, 1571- 1577.
5
Decaens, C., Bara, J.. Rosa, B., Daher, N. and Burtin. P. (1983) Early oncofetal antigenic modifications during rat colonic carcinogenesis. Cancer Res., 43, 355 - 362. Decaens, C., Gautier, R.. Daher, N., Bara, J. and Burtin, P. (1989) Induction of rat intestinal carcinogenesis with single doses, low and high repeated doses of 1,2dimethylhydrazine. Carcinogenesis., 10. 69 - 72. Deschner, E.E. (1974) Experimentally induced cancer of
6
7 8
9
10
11
12
. The
13
14 15
16
References Berenblum, I., Haran, N. and Rosin, A. (1956) The carcinogenic action in the mouse of 20-methylcholanthrene by rectal administration. Am. J. Pathol., 32, 579 - 589. Bralow, S.P. and Weisburger, J.H. (1976) Experimental carcinogenesis in the digestive organs. Clin. Gastroenterol.. 5. 527-530. Cowdry, E.V (1968) Malignant neoplasm of digestive organs and peritoneum, Large intestine. in: Etiology and Prevention of Cancer in Man, pp. 74-94. Editor: E.V. Cowdry. Appleton-Century Crofts, New York.
17
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colon. Cancer, 34, 824-828. Druckrey. H., Preussmann, R., Matzkies, F. and Ivankovie. S. (1967) Selektive Erzeugung Von Darmkrebs bei Ratten durch 1.2-dimethylhydrazine. Naturwissenschaften. 54, 285 - 286. Druckrey, H. (1970) Production of colonic carcinomas by l-2 dialkylhydrazines and azoxyalkanes. In: Carcinoma of the Colon and Antecedent Epithelium, pp. 267 -279. Editor: W.J. Burdette. Thomas, Springfield, U.S.A. Filipe, M.I. (1975) Mucous secretion in rat colonic mucosa during carcinogenesis induced by dimethylhydrazine. A morphological and histochemical study. Br. J. Cancer, 32, 60-77. Graham, R.C.. Lundholm, U. and Karnovsky, M. (1965) Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J. Histochem. Cytochem., 13. 150- 152. Hoch-Ligeti, C.. Argus, M.E. and Arcos, J.C. (1968) Combined carcinogenic effects of dimethylnitrosamine and 3-methylcholanthrene in the rat. J. Natl. Cancer Inst., 40. 535 - 549. Hornburger, F.. Hsuch, S.S., Kerr, C.S. and Russfield, A.B. (1972) Inherited susceptibility of inbred strains of Syrian hamsters by induction of subcutaneous sarcomas and mammary and gastrointestinal carcinomas by subcutaneous and gastric administration of polynuclear hydrocarbons. Cancer Res., 32, 360 - 366. Horava. A. and Haam. E.V. (1959) Experimental carcinoma of the colon. Cancer Res.. 18, 764- 767. Huggins. C. and Lorraine, C.G. (1963) Sarcoma induced remotely in rats fed 3-methylcholanthrene. Cancer Res., 23. 477 - 480. Ishioka, T., Kuwabara, N. and Fukuda, Y. (1985) Induction of colorectal adenocarcinoma in rats by amylopectin sulfate. Cancer Lett., 26, 277 - 282. Kouri. R.E.. Ratrie, H. and Whitmore. C.E. (1973) Evidence of genetic relationship between susceptibility to 3-methylcholanthrene induced subcutaneous tumours and inducibility of aryl hydrocarbon hydroxylase. J. Natl. Cancer Inst.. 51. 197-200. Laqueur. G.L.. Michaelson, 0.. Whiting. M.G. and Kurland, L.T. (1963) Carcinogenic properties of nuts from cycas circinalis. J. Natl. Cancer Inst., 31. 919 -933. Laqueur, G.L. (1965) The induction of intestinal neoplasms in rats with the glucoside cycasin and its
183
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aglycone. Virchows Arch. (Path of Anat.), 340, 151- 163. Morodomi, T.. Isomoto. H., Shirousu, G., Shirouzu, K.. Kakegawa, T., Irie, K., and Morimatsu, M. (1989) Effect of OK-432 on large bowel carcinogenesis in rats. Dis. Colon Rectum, 32, 871-877. Narisawa, T., Sate, T., Hayakawa, M., Sakuma, A. and
22
Nakamo, H. (1971) Carcinoma of the colon and rectum of rats by rectal infusion of N-methyl-N ‘-nitro-N-nitrosoguanidine. Jpn. J. Cancer Res., 62, 231-234. Narisawa. T. and Nakano, H. (1973) Carcinoma of the
20
23
large intestine of rats induced by rectal infusion of methylazoxymethanol. Jpn. J. Cancer Res., 64, 93 - 95. Rack, F.J.. Kaufman, N. and Simeone, P.A. (1955) Experimental carcinogenesis. The effect of 20-methylcholanthrene applied directly Res., 15. 722- 725.
to the colon
of mice.
Cancer
24
25
26
27
Reddy, Wynder,
B.S., Weisburger, E.L. (1974) Colon
J.H., Narisawa. T. and carcinogenesis in germ free
rats with 1,2-dimethylhydrazine and N-methyl-N ’ nitro-Nnitrosoguanidine. Cancer Res., 34, 2368 - 2372. Shirai, T., Nakanowatari, J., Kurata, Y., Fukushima, S. and Nobuyuki, I. (1983) Different dose-response relationships in the induction of different types of colonic tumours in Wistar rats by 1,2-dimethylhydrazine. Jpn. J. Cancer Res., 74, 21-27. Tarin, D. (1972) Morphological studies on the mechanism of carcinogenesis. In: Tissue Interactions in Carcinogenesis, pp. 227-289. Editor: D. Tarin. Academic Press, London and N.Y. Thurnherr, N., Deschner, E.E., Stonehill. E.H. and Lipkin, M. (1973) Induction of adenocarcinomas of the colon in mice by weekly injection of 1,2-dimethylhydrazine. Cancer Res., 33, 940- 945.
177
Induction of colorectal cancer in rats by ZO-methylcholanthrene R.N. Baral and P. Maity Department of Cell Biology, Chittoranjon (Received (Accepted
National
Cancer
Institute, 37, S.P. Mukherjee
700 026 (India)
Colorectal
Introduction carcinoma
was
induced
Sprague - Dawley rats by 20-methylcholanthrene. Macroscopical studies revealed that tumors, either sessile type or semi-peduncul-
in
Colorectal carcinoma has been induced in mice and rats by different carcinogenic agents, like cycasin, methylazoxymethanol (MAM) (DMH) [8,9], [l&19], 1,2-d imethylhydrazine IV-methyl-N’nitro-IV-nitrosoguanidine (MNNG) [Zl], N-methyl-N-nitrosourea (MNU) [20] etc. and in most of the cases, appearance of the carcinoma were reported within 25 weeks of the treatment [2,8,9,16,18 - 211. 20-Methylcholanthrene (MCA) was considered as a potent cancer causing agent [12,17,26]. Sarcoma in rats produced by MCA was described by Huggins et al. in 1963 [15]. Homburger et al., in their elaborate survey of chemical carcinogenesis found that oral administration of MCA in hamsters causes colon cancer [ 131. Carcinogenic effects of 20-MCA in the mice large intestine were also reported [1,23]. In 1958, Horava et al. described cecoappendiceal carcinoma in rats by local application of the 20-MCA, however, no tumors were found by them in the colons of experimental animals [ 141. In the present investigation, we intend to examine, whether colorectal cancer can be produced in rats by direct application of MCA through the rectum.
the
ated polyp, were generally observed after 32 weeks of the carcinogen treatment. In the distal colon 46.9% tumors appeared, whereas
20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively. Sequential histiopathological studies indicated that hyperplaria of goblet cells was common in early stages, which was reduced thereafter. Carcinogenesis progressed with the appearance of the different grades of dysplasia in colmucosa dysplasia
wifh first incidence of the in rats at the 20th week and
carcinoma at the close of 28th week. of the carcinomas were multifocal in origin and were well differentiated adenocarcinema with primary invasion at the submucosa. In immunohistological studies, this carcinoma was also reactive with monoclonal in situ Most
antibody carcinoma
660, prepared against a co/ore&al associated mucin antigen.
colorectal Keywords: methylcholanthrene; tumors;
carcinogenesis; adenocarcinoma
Materials Correspondence to: P. Maity, Department of Cell Biology, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Calcutta
Calcutta
19 September 1991) 1 October 1991)
Summary
ore&al severe
Road.
Carcinogenesis
700 026, India.
0304.3835/92/$05.00 Printed and Published
G 1992 Elsevier Scientific Publishers in Ireland
and Methods
Seventy male Sprague - Dawley rats, 8 weeks Ireland
Ltd
178
of age, weighing approximately 130 g at the beginning of the experiment, were selected for this study and randomly divided into three groups. A group of 40 rats received MCA dissolved in benzene, another group of 20 rats was treated with benzene alone and 10 rats were maintained as normal control. For colorectal carcinogenesis, each rat received 2 mg MCA per week. Cotton threads soaked with 0.5% MCA solution (1 mg MCA + 200 ~1 benzene) were placed into the lumen of the large intestine through anal orifices of the experimental rats and carcinogen was inserted twice a week. A group of animals received benzene only in the same way as the carcinogen-treated group. At a regular interval of 4 weeks, rats from each group (5 from MCA + benzene; 2 from benzene alone and 1 from normal) were sacrificed for histological study. The experiment was continued up to 32 weeks. Histology The colon and rectum of animals that had been treated with MCA for different periods were opened, cleaned in phosphate buffered saline (PBS), pH 7.2, and examined for the size and distribution of tumors. number, Thereafter, the rectum, distal and proximal colon of control as well as experimental animals were fixed in 95% ethanol [5] and coiled up into rolls [lo]. Tissues were processed by routine histological methods and sections were prepared. Each section was stained with haematoxylin-eosin and also studied immunohistochemically. Histopathological staging Preneoplastic and neoplastic lesions were classified on the basis of histological differentiation according to the method described by M.I. Filipe [lo] and Decaens et al. [5]. lmmunohistochemistry A monoclonal antibody (MAb 660), against a mucin-associated 660 antigen, was used in this experiment. MA\, 660 was received as a gift from Dr. C. Decaens of Mucin Immunochemistry Laboratory, Villejuif, Cedex, France.
Sections were deparaffinised in toluene and endogeneous peroxidase activity was inhibited by incubation of sections with 0.5% H202 in methanol for 20 min. After thorough washing in PBS, tissue sections were incubated overnight at 4OC with MAb 660. Incubated sections, after washing with PBS, were incubated again with sheep antiserum against mouse IgG (H + L) labelled with peroxidase (Diagnostics Pasteur, Marnes La Coquette, France) at a l/100 dilution for 45 min. The sections were washed with PBS and peroxidase activity was revealed using aminoethylcarbazol according to the method of Graham et al. [ll]. Slides were counter stained with haematoxylin before microscopic examination. Statistics The Scheffe’s F-test was used to determine the significance of differences between the number of tumors in the rectum, distal and proximal colon. Results Macroscopy The gross appearance of the luminal surface of the distal colon was morphologically abnormal in the late stages of the carcinogenesis, but very few animals possessed tumorous growth before 32nd week of the treatment. All rats sacrificed after 32nd week showed the presence of tumors within the large intestinal lumen (Table I). Most of the growths (Fig. l), found in the colorectum, were of the sessile type with a smooth surface structure, but a few tumors were semipedunculated polyps and a single cauliflower-like growth was observed in the rectum of one induced animal. Histopathology The histopathologic findings are shown in Table II. Mucosal changes following MCA administration had been classified into hyperplastic foci, different grades of dysplasia and carcinoma. Hyperplasia Epithelial
hyperplasia
was
observed
in
179
Table 1. Distribution, size and number of tumors appeared in the colorectum of five rats treated with MCA for 32 weeks. Sites
Tumor size (mm)
Number of tumors
Rectum
55 5.1-9.9 2 10 55 5.1-9.9 2 10 55 5.1-9.9 2 10
3 4 3 8 9 6 4 7 5
Distal colon
Proximal colon
‘Significantly
different
from rectum,
Total number of tumors (%)
10 (20.4)
23’
(46.9)
16 (32.6)
P < 0.01.
animals treated with MCA for 4- 12 weeks. The colorectal tissues of these animals showed dilated crypts due to marked increase in the secreting goblet cells. mucous A few hyperplastic glands were mildly hyperchromatic in animals that had received MCA for more than 8 weeks, but the nuclei were aligned regularly on the basement membrane.
Fig. 1.
Macroscopic
tumors
appeared
Dysplasia divided into different Dysplasias were groups according to the severity of the disease, i.e. from grade 0 to grade 3. Rats received carcinogen not more than 16 weeks, were included in the group of dysplasia grade 0. In this grade, crypts may be dilated, but a significant reduction in the number of goblet cells were seen. This reduction in the cellular activity of the goblet cells was progressed with the advancement of the disease. Animals after 16 weeks of carcinogenesis showed features of Dysplasia grade 1, like cellular atypia, however, nuclear polarity was unchanged in these animals. In the 24th and 28th week, animals showed irregular tubular structure, with loss of nuclear polarity and appearance of the mitotic figures (Dysplasia grade 2). Animals that received more than 28 weeks treatment possessed dysplastic glands of the third grade. Back-to-back architecture along with severe glandular atypia were seen in most of these animals. Carcinoma
The malignant neoplasms observed in our experiments generally after 32 weeks of carcinogen insertion, were either in situ or in(Fig. 2). These are filtrating carcinomas
at distal (D) and proximal
(P) colon of rat treated
with MCA for 32 weeks.
180
Table II. Time of appearance Weeks of treatment
4 8 12 16 20 24 28 32
Features
of different
carcinogenic
x
carcinogenesis.
Goblet cellular proliferation
Hyperchromatism
Atypism
Loss of polarity
Expression of mitotic figures
+++ +++ +++ ++ ++ f f -
+ + + + +/+ ++ +++ +++ +++
Not observed do do +/+ + ++ +++ +++ +++
Not observed do do + ++ +++ +++ +++
As normal do do ++ ++ +++ +++ +++
and marked
exp:ession
characterized by loss of glandular architecture with different degrees of differentiation. Most of the carcinomas were multifocal in origin and were well-differentiated adenocarcinoma, although a few mucosal foci with moderate or poor differentiation were also demonstrated. Carcinomas often reached a depth of submucosa, but only two incidents were demonstrated where carcinomas invaded to muscular layer. Areas of hyperplasia and dysplasias were
Fig. 2.
during colorectal
of carcinogenesis
+ , + + , + + + = mild, moderate
Eosin,
features
An ulcerated 120.
adenocarcinoma
at the rat rectum
of different
carcinogenic
features.
often adjacent to the carcinoma invasive carcinomas.
in situ and
Controls The changes observed in rats treated with benzene alone were not comparable with the MCA treated groups. The only change that appeared in the mucosa was occasional mild glandular hyperplasia. The pattern of crypts were completely unaltered. The group of ten animals used as normal
after 32 weeks of the MCA treatment.
Haematoxylin
and
181
control showed neither macroscopical growth nor microscopical lesions in the colorectum. lmmunohistochemistry
The study with MAb 660 was performed on the colorectal mucosae of normal and MCA treated rats. Most of the large intestinal carcinomas appeared in our studies were reactive with MAb 660. This antigen was also expressed in dysplastic (Fig. 3)) hyperplastic as well as apparently normal mucosal glands of MCA induced animals. However, MAb 660 reactivity was absent in colorectal mucosae of normal rats. Discussion
To our knowledge, we are the first to report the induction of colorectal carcinoma in Sprague - Dawley rats by direct application of MCA through the rectum. Although Horava et al. tried to develop tumors at colon by the
Fig. 3. A dysplastic gland of distal colon, adjacent to carcinoma. exhibiting 660 antigen, immunoperoxidase. x480.
same carcinogen, but they were not successful in that attempt [14]. Macroscopical studies that revealed the incidence of tumors in 100% of the rats occurred only after 32 weeks of treatment. M.I. Filipe reported macroscopical lesions at the colon after 19 weeks in all the DMH-treated rats [lo]. Narisawa demonstrated the same within 25 weeks during carcinogenesis with MAM [22]. In comparison, the animal system described here, took longer time to produce tumorous growth. This slow progression of the disease may not be due to low dose of the MCA. An animal, undergoing treatment for 32 weeks, received a total dose of 64 mg of MCA and this dose was comparatively higher than the dose described in DMH, MAM, MNNG and MNU induced systems [6,7,20,24,25]. The maximum number of tumors (46.9%) appeared in the distal colon after 32 weeks of MCA instillation, whereas 20.4% and 32.6% tumors were found in the rectum and proximal colon, respectively (Table I). Narisawa et al. also induced many tumors in the distal large bowel of rats by the intrarectal administration of MNNG and MAM [21,22]. This part of the colon was also described as a most sensitive site of occurrence of malignant growth in other experimental models [7,10,25]. Different features of carcinogenesis in relation to duration of treatment are presented in Table II. The earlier change, detected after 4 weeks, was cellular hyperplasia of goblet cells, which was reduced linearly with the progression of the disease. This observation correlates well with the features reported in a similar stepwise investigation carried out by Thurnherr et al. [27] and M.I. Filipe [lo]. Severe dysplasia and in situ carcinoma were observed after 13th week and 15th week respectively during DMH induced carcinogenesis [lo], whereas, in the present study, severe dysplasia was first seen in an animal at the 20th week and in situ carcinoma at the end of the 28th week. The malignant neoplasms produced in our experiments were adenocarcinoma with difof differentiation. Several ferent degrees groups of investigators reported this type of
182
carcinoma in their experimental system during carcinogenesis with a variety of carcinogens [7,10,16,20,22]. In human system also, is the most common adenocarcinoma malignancy of the large gut [3]. MCA-induced carcinoma invaded mainly to the submucosa and this finding was similar to the nature of observed in other experimental invasion models [10,20,22]. The expression of an oncofetal mucin antigen, the 660 antigen, in the preneoplastic as well as neoplastic colorectal mucosae during MCA-induced carcinogenesis was similar to the results described by Decaens et al. [4] and this observation provides an additional support in favour of carcinogenic changes produced by MCA. It may be concluded from our results that a new experimental model of slow progressing colorectal carcinoma has been developed in rats by applying MCA locally and this model may help us in the understanding of the mechanism of carcinogenesis in human beings, where it is a long-term process. Acknowledgements authors thank Dr. J. Roychowdhury, Director, CNCI, Calcutta, for providing all facilities. We wish to express our thanks to Dr. R.N. Chakraborty for his helpful suggestions in histopathological studies. We are also indebted to Dr. C. Decaens for her gift of the monoclonal antibody. The first author gratefully acknowledges the financial assistance given by Indian Council of Medical Research, New Delhi.
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Decaens, C., Gautier, R., Bara, J., Daher, N.. Pendu, J.L. and Burtin, P. (1988) A new mucin associated oncofetal antigen, a marker of early carcinogenesis in rat colon. Cancer Res., 48, 1571- 1577.
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Decaens, C., Bara, J.. Rosa, B., Daher, N. and Burtin. P. (1983) Early oncofetal antigenic modifications during rat colonic carcinogenesis. Cancer Res., 43, 355 - 362. Decaens, C., Gautier, R.. Daher, N., Bara, J. and Burtin, P. (1989) Induction of rat intestinal carcinogenesis with single doses, low and high repeated doses of 1,2dimethylhydrazine. Carcinogenesis., 10. 69 - 72. Deschner, E.E. (1974) Experimentally induced cancer of
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