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Dose response of 1,2-dimethylhydrazine and methylazoxymethanol acetate in the F 344 rat
Cancer Letters,
8 (1980)
o EisevierfNorth-Holland
271-278 Scientific Publishers Ltd.
271
DOSE RESPONSE OF 1,2-DIMETHYLHYDRAZINE AND METHYLAZOXYMETHANOL ACETATE IN THE F 344 RAT
ERNEST
E. McCONNELLa,
R.E. WILSONa,
J.A. MOOREa
and J.K. HASEMANb
aEnuironmental Biology Branch and bBiometry Branch, National Institute of Enuironmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) (Received 26 June 1979) (Accepted 23 August 1979)
SUMMARY
Weanling male and female F 344 rats were given various doses of 1,2dimethylhydrazine (DMH) or methylazoxymethyl acetate (MAMAc), and were then held for 46-64 weeks in an effort to determine a dose response and establish a dose level which would produce a low level of intestinal neoplasia with a minimal number of tumors in other organs. DMH proved superior to MAM in this respect, although liver lesions were still observed with both compounds at the lowest carcinogenic intestinal dose.
INTRODUCTION
The use of natural cycasin [6] and synthetic chemicals [8] for the production of colon cancer in rats has been known for several years. The most popular chemicals for this purpose are 1,2_dimethylhydrazine (DMH) [3], methylazoxymethanol (MAM) or MAM acetate (MAMAc) [5] and azoxymethane (AOM) [ll].While DMH and AOM require metabolic change within the host to become carcinogenic, MAM appears to be proximate carcinogen 1141. Recently it has been shown that there is epidemiological evidence of increased rates of colon and rectal cancer among workers exposed to asbestos [ 131. In pursuing the possible carcinogenic potential of orally ingested asbestos (chrysotile) in rats, it was felt that it was necessary to also investigate its potential as a promoter of intestinal cancer. To accomplish this task, a model compound was needed which would produce a relatively low incidence (15 + 5%) of intestinal cancer (hopefully restricted to the colon) and relatively little neoplasia at other sites in the body. Since little dose response data was available in this regard, we designed the following experiment using various levels of DMH and MAM.
272 MATERIALS
AND METHODS
Animals Male and female specific pathogen free F 344 rats (Charles River, Wilmington, MA), 6 weeks old, were divided into various sized groups (Table 1) in order to place more animals in groups where a lower tumor incidence was suspected. Chemicals DMHa2Cl or MAMAc (Aldrich Chemical Co., Milwaukee, WI) were dissolved in double distilled water, buffered (acetate) to pH 5.0, placed on ice, and were administered within 4 h of preparation. Treatment Each rat was given 5 doses of either DMH or MAMAc via gavage at 2-week intervals. The rats were housed 3 per polycarbonate cage (45 X 20 X 20 cm) and given food (NIH ‘31’ autoclavable lab blocks, Ziegler Bros., Gardners, PA) and water ad libitum. The rats were weighed every 2 weeks for 6 months and then once per month for the duration of the study (14 months) when all surviving rats were sacrificed. Pathology examination At necropsy the length of the entire digestive tract was measured, opened, washed with physiological saline and laid flat (mucosal surface up) on a piece of moistened, clear plexiglass. Grossly visible or suspect tumors were recorded as to location, size and macroscopic morphology. Other tissues routinely collected for histopathologic examination included : stomach, 3 areas of small intestine, cecum, entire colon (French roll), kidneys, liver mesenteric, lymph nodes and Zymbals gland. Lesions in other organs were collected when encountered. All tissues were routinely fixed in neutral buffered 10% formalin, embedded in paraffin, sectioned at 6 pm and stained with hematoxylin and eosin. Statistics The multi-stage model for cancer [1,4] was assumed to hold for the colon tumor data. Dose-response curves were estimated using the computer program described by Crump et al. [2]. The dose curves were not used for extrapolation (i.e., to estimate risk at very low doses), but rather to estimate the dose level within the experimental range that would produce a low incidence (lo-15%) of intestinal neoplasia. For this purpose alternative models (Mantel-Bryan Log-Probit [7] or Gamma Multi-Hit [12] ) would have given quite similar results. RESULTS
The only overt sign of toxicity was a mild but significant (P = < 0.01)
M M M M M M M F F F F F F F
30 15 7.5 4. 1 0.2 0 30 15 7.5 4 1 0.2 0
18 24 27 27 27 27 30 18 24 27 27 27 27 30
N
16 12 8 2 0 0 0 12 6 3 1 0 0 1
Total rats with tumors
aNumber of rats with tumors. bAdditional tumor in cecum.
Sex
Dose mg/kg x5
14 (78) 10 (42) 5 (18) 2(7) O(0) O(0) O(0) 11 (61) 5 (21) l(4) O(0) O(0) O(0) O(0)
GI tumors (%)
0 0 0 0 0 0 0 0 0 0 0 0 0 0
Stomach
0 1 0 0 0 0 0 0 0 0 0 0 0 0
Small intestine
TUMOR INCIDENCEa IN F 344 RATS TREATED WITH DMH
TABLE 1
Bb 5 2 0 0 0 11 5 1 0 0 0 0
.14
Colon
8 3 1 0 0 0 0 2 0 0 1 0 0 0
Zymbal’s gland
0 0 0 0 0 0 0 2 0 0 0 0 0 0
Kidney
1 2 1 3 1 0 1 4 10 3 1 0 1 2
Foci cell alteration
Liver
0 0 1 0 0 0 0 0 1 1 0 0 0 0
Neoplastic nodule 0 0 0 0 0 0 0 0 0 1 0 0 0 0
‘_
44 50 52 55 59 61 64 44 49 53 56 60 62 64
Carcinoma Weeks at sac.
274
persistant d&rease in the rate of body weight gain after 4 weeks exposure to the highest dose (30 mg/kg) of DMH in males and after 3 weeks in females treated with MAMAc. Several months later, as neoplasia became evident with associated weight loss, males and females exposed to both compounds at the 30 mg/kg dose showed less weight gain. There was a dose response with regard to the total number of tumor bearing rats, organ location and latent period of the tumors induced by both DMH and MAMAc (Table 1 and 2). The first tumors were observed at 22 weeks in the colon of 2 male rats which died after being exposed to DMH (one each 30 and 15 mg/kg). The first tumors (colon) in rats exposed to MAMAc (30 mg/kg) (1 male and 1 female) were found at 26 weeks. Several other animals in the higher dose groups of both chemicals died or were killed in a moribund state because of neoplasia prior to the termination of the study. Tumors were observed most commonly in the gastrointestinal tract, Zymbal’s gland, kidney, liver and mammary gland and occasionally up to 4 different organs showed neoplasia within the same animal (Tables 1 and 2). In the lower dose groups the tumors were confined almost entirely to the colon. Stomach All tumors observed in the stomach were in rats exposed to MAMAc (Table 2). They were confined to the non-glandular portion (forestomach) and varied from small squamous papillomas (9 rats) to invasive squamous cell carcinomas (6 rats). No metastases were observed. Small intestine Tumors of the small intestine were rare, but were found with both DMH and MAMAc (30 and 15 mg/kg, male only). All tumors were epithelial in origin and most appeared to arise in the middle portion of the small intestine in or near a lymphoid (Peyer’s) patch. Large intestine Tumors of the large intestine were more frequently observed in males than females at the same dose and DMH caused tumors at a lower dose than MAMAc (Tables 1 and 2). The location was the same with both compounds with a majority located in the proximal or distal portions of the colon. Only one neoplasm was found in the cecum and none in the anus. Most of the rats had fewer than 3 tumors in the colon but occasional animals had up to 10. All tumors of the colon were of an epithelial type and varied from carcinoma in situ to small well differentiated adenomas, polypoid carcinoma, tubular adenocarcinoma and mutinous (signet ring) carcinoma. The mutinous type often metastasized regardless of the dose of the carcinogen or sex of the animal; most frequently to the regional lymph nodes and on the serosal surface of adjacent viscera. Again, many of the smaller tumors appeared to arise in the area of a lymphoid patch, especially in the proximal colon. With one possible exception (male rat-MAMAc) the multistage model was found to provide an excellent fit to the observed tumor rates (Figs. 1 and 2).
M M M M M M M F F F F F F F
30 15 7.5 4 1 0.2 0 30 15 7.5 4 1 0.2 0
18 24 25 27 26 27 30 18 24 27 27 27 27 30
N
16 12 4 0 0 1 1 11 6 1 2 0 0 6
Total rats with tumors
aNumber of rata with tumors.
Sex
Dose mg/kg x5
12 (67) 11 (46) 2( S) 0( 0) 0( 0) 0( 0) 0( 0) 6 (44) 5 (21) 1( 4) 0( 0) 0( 0) 0( 0) 0( 0)
GI tumors
5 3 0 0 0 0 0 4 3 0 0 0 0 0
Stomach
2 0 0 0 0 0 0 0 0 0 0 0 0 0
Small intestine
10 10 2 0 0 0 0 6 2 1 0 0 0 0
Colon
TUMOR INCIDENCEa IN F 344 RATS TREATED WITH MAMAc
TABLE 2
6 0 0 0 0 0 0 0 0 0 1 0 0 0
Zymbal’s gland
6 0 0 1 0 0 0
0 0 0 0 0 0 0
Kidney
6 16 5 4 1 1 1 5 10 7 1 0 0 2
Foci cell alteration
Liver
0 1 0 0 0 0 0 2 0 0 1 0 0 0
Neoplastic nodule
0 0 0 0 0 0 0 0 0 0 0 0 0 0
46 52 56 59 62 64 64 46 53 55 54 61 63 64
Carcinoma Weeks at sac.
ul
Y
276 80-
70-
+ Male DMH O--Female DMH
0
5
IO
Fig. 1. Dose response
15 Dose
20 25 mg /kg x 5
curves for colon
30
tumors of male and female
rats exposed
to DMH
609 ? 8 50E 0 ‘Z 402
O-Male MAM o---Female MAM
Dose- mg/ kg x 5 Fig. 2. Dose response MAMAc.
curves for colon
tumors
of male and female
rats exposed
to
277
Zymbal gland
Tumors of the Zymbal gland were usually unilateral and often presented as a fungating or caseous mass at the base of the ear. They were more common in males than females. Histologically, they all resembled a squamous cell carcinoma with various degrees of anaplasia. Necrosis and inflammatory response often obscurred the tumor. No evidence of metastasis was observed. Preputial gland
The preputial gland tumors were observed in MAMAc (one 15 mg and one 7.5 mg) treated rats only. They appeared as small, hard nodules which histologically resembled the Zymbal gland tumors. Kidney
Tumors of the kidney were all found in female rats and more so with MAM than DMH. They were unilateral and varied from small cortical foci to highly invasive tumors composed of spindle shaped cells which involved most of the organ. No evidence of metastasis was found. Liver
Hepatocellular carcinoma and neoplastic nodules [ 151 were encountered rarely, and the former only in the rats exposed to MAMAc. However, foci of cellular alteration were noted at the higher doses of both compounds, but more so with MAMAc (Tables 1 and 2). They were composed of basophilic or eosinophilic or mixed-cell types [ 151. Mammary
gland
Mammary gland tumors were encountered with low frequency at the higher doses of MAMAc, in both male (one 7.5 mg) and female (one 30 mg and one 15 mg) animals. One similar tumor was observed in a female control. Microscopically, all were similar and were classified as cystic, fibroadenomas. DISCUSSION
The objective of this study was to obtain a dose of either DMH or MAMAc which would produce a relatively low incidence of neoplasia of the large intestine while at the same time producing little toxicity or neoplasia at other sites in the body. While a great deal of information is available on the morphology [9] of tumors produced by these compounds none could be found which showed a dose response relationship. However, subsequent to the initiation of this study a dose response experiment using a single subcutaneous (s.c.) dose of AOM in male Fischer rats has been reported [16]. The author found that a single S.C.injection of 0.8 mg/kg of AOM produced an incidence of 20% intestinal neoplasia, all of which was restricted to the large intestine with no evidence of neoplasia at other sites. Another recent study [lo] demonstrated that dosing sequence affects the target site. Daily S.C.injections of DMH in rats produced tumors in the large intestine only, while
278
larger doses given once to twice weekly stimulated tumors in various segments of the gastrointestinal tract (total dose was approximately equal). Both DMH and MAMAc showed a dose response relationship with a greater dose being required to produce intestinal tumors in female than male rats. In addition, DMH produced more intestinal tumors at lower doses than MAMAc. Importantly, as the dose of either compound was reduced, the incidence of tumors at other sites (Zymbal’s gland, kidney, liver) also decreased. ACKNOWLEDGEMENT
We thank Mr. John Fawkes, Environmental Chemistry Branch, NIEHS, for his efforts to analyze the chemicals for purity and potency. REFERENCES 1 Armitage, P. and Doss, R. (1961) Stochastic models for carcinogenesis. In: Proceedings of the 4th Berkeley Symposium on Mathematical Statistics and Probability, Vol. 4, pp. 19-38. University of California Press, Berkeley, California. 2 Crump. K.S., Guess, H.A. and Deal, K.L. (1977) Confidence intervals and test of hypotheses concerning dose response relations inferred from animal carcinogenicity data. Biometrics, 33, 437-451. 3 Druckrey, H., Preussmann, R., Matzkies, F. and Ivankovic, S. (1967) Selektive erzeugung von darmkrebs bei ratten durch 1,2_dimethylhydrazine. Naturwissenschaften, 54,285-286. Guess, H.A., Crump, K.S. and Peto, R. (1977) Uncertainty estimates for low-dose-rate extrapolation of animal carcinogenicity data. Cancer Res., 37, 3475-3483. Laqueur, G.L. and Matsumoto, H. (1966) Neoplasms in female Fischer rats following injection of methylazoxymethanol. J. Natl. Cancer Inst., 37, 217-232. Laqueur, G.L. and Spatz, M. (1968) Toxicology of cycasin. Cancer Res., 28, 22622267. Mantel, N., Bohidar, N.R., Brown, D.C., Ciminera, J.L. and Tukey, J.W. (1971) An improved “Mantel-Bryan” procedure for “safety testing” of carcinogens. Cancer Res., 35,865-872. 8 Pozharisski, K.M. (1973) Tumors of the intestines. In: Pathology of Tumors in Laboratory Animals, pp. 119-130. Editor: V.S. Jubusov. IARC Scientific Publication No. 5, WHO Lyon, France. 9 Pozharisski, K.M. (1975) Morphology and morphogenesis of experimental epithelial tumors of the intestine. J. Natl. Cancer Inst., 54,1115-1135. 10 Pozharisski, K.M., Vokhmianin, V.N., Kulakov, N.A., Balanski, R.M., Likhachev, A&., Petrov, AS. and Shaposhnikov, 1a.D. (1978) Effects of schedule of 1,2-dimethylhydrazine administration on its carcinogenicity. Bull. Eksp. Biol. Med., 85, 337-340. 11 Preussmann, R., Druckrey, H., Ivanovic, S. and Hodenberg, A.V. (1969) Chemical structure and carcinogenicity of aliphatic hydrazo, azo, and azoxy compounds and of triazenes, potential in vivo alkylating agents. Ann. NY Acad. Sci., 163, 697-716. 12 Rai, K. and Van Ryzin, J. (1979) Risk assessment of toxic environmental substances using a generalized multi-hit dose response model. In : Energy and Health, pp. 99-l 17. Editors: N. Breslow and A. Whittemore, Siam Press, Philadelphia, PA. Rev. Fr. Mal. 13 Selikoff, I.J. (1976) Asbestos disease in the United States 1918-1975. Resp., 4, 7-24. 14 Spatz, M., McDaniel, E.G. and Laqueur, G.L. (1966) Cycasin excretion in conventional and germfree rats. Proc. Sot. Exp. Biol. Med., 121,417-422. 15 Squire, R.A. and Levitt, M.L. (1975) Report of a workshop on classification of specific hepatocellular lesions in rats. Cancer Res., 35, 3214-3223. 16 Ward, J.M. (1975) Dose response to a single injection of azoxymethane in rats. Vet. Pathol., 12, 165-177.
8 (1980)
o EisevierfNorth-Holland
271-278 Scientific Publishers Ltd.
271
DOSE RESPONSE OF 1,2-DIMETHYLHYDRAZINE AND METHYLAZOXYMETHANOL ACETATE IN THE F 344 RAT
ERNEST
E. McCONNELLa,
R.E. WILSONa,
J.A. MOOREa
and J.K. HASEMANb
aEnuironmental Biology Branch and bBiometry Branch, National Institute of Enuironmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) (Received 26 June 1979) (Accepted 23 August 1979)
SUMMARY
Weanling male and female F 344 rats were given various doses of 1,2dimethylhydrazine (DMH) or methylazoxymethyl acetate (MAMAc), and were then held for 46-64 weeks in an effort to determine a dose response and establish a dose level which would produce a low level of intestinal neoplasia with a minimal number of tumors in other organs. DMH proved superior to MAM in this respect, although liver lesions were still observed with both compounds at the lowest carcinogenic intestinal dose.
INTRODUCTION
The use of natural cycasin [6] and synthetic chemicals [8] for the production of colon cancer in rats has been known for several years. The most popular chemicals for this purpose are 1,2_dimethylhydrazine (DMH) [3], methylazoxymethanol (MAM) or MAM acetate (MAMAc) [5] and azoxymethane (AOM) [ll].While DMH and AOM require metabolic change within the host to become carcinogenic, MAM appears to be proximate carcinogen 1141. Recently it has been shown that there is epidemiological evidence of increased rates of colon and rectal cancer among workers exposed to asbestos [ 131. In pursuing the possible carcinogenic potential of orally ingested asbestos (chrysotile) in rats, it was felt that it was necessary to also investigate its potential as a promoter of intestinal cancer. To accomplish this task, a model compound was needed which would produce a relatively low incidence (15 + 5%) of intestinal cancer (hopefully restricted to the colon) and relatively little neoplasia at other sites in the body. Since little dose response data was available in this regard, we designed the following experiment using various levels of DMH and MAM.
272 MATERIALS
AND METHODS
Animals Male and female specific pathogen free F 344 rats (Charles River, Wilmington, MA), 6 weeks old, were divided into various sized groups (Table 1) in order to place more animals in groups where a lower tumor incidence was suspected. Chemicals DMHa2Cl or MAMAc (Aldrich Chemical Co., Milwaukee, WI) were dissolved in double distilled water, buffered (acetate) to pH 5.0, placed on ice, and were administered within 4 h of preparation. Treatment Each rat was given 5 doses of either DMH or MAMAc via gavage at 2-week intervals. The rats were housed 3 per polycarbonate cage (45 X 20 X 20 cm) and given food (NIH ‘31’ autoclavable lab blocks, Ziegler Bros., Gardners, PA) and water ad libitum. The rats were weighed every 2 weeks for 6 months and then once per month for the duration of the study (14 months) when all surviving rats were sacrificed. Pathology examination At necropsy the length of the entire digestive tract was measured, opened, washed with physiological saline and laid flat (mucosal surface up) on a piece of moistened, clear plexiglass. Grossly visible or suspect tumors were recorded as to location, size and macroscopic morphology. Other tissues routinely collected for histopathologic examination included : stomach, 3 areas of small intestine, cecum, entire colon (French roll), kidneys, liver mesenteric, lymph nodes and Zymbals gland. Lesions in other organs were collected when encountered. All tissues were routinely fixed in neutral buffered 10% formalin, embedded in paraffin, sectioned at 6 pm and stained with hematoxylin and eosin. Statistics The multi-stage model for cancer [1,4] was assumed to hold for the colon tumor data. Dose-response curves were estimated using the computer program described by Crump et al. [2]. The dose curves were not used for extrapolation (i.e., to estimate risk at very low doses), but rather to estimate the dose level within the experimental range that would produce a low incidence (lo-15%) of intestinal neoplasia. For this purpose alternative models (Mantel-Bryan Log-Probit [7] or Gamma Multi-Hit [12] ) would have given quite similar results. RESULTS
The only overt sign of toxicity was a mild but significant (P = < 0.01)
M M M M M M M F F F F F F F
30 15 7.5 4. 1 0.2 0 30 15 7.5 4 1 0.2 0
18 24 27 27 27 27 30 18 24 27 27 27 27 30
N
16 12 8 2 0 0 0 12 6 3 1 0 0 1
Total rats with tumors
aNumber of rats with tumors. bAdditional tumor in cecum.
Sex
Dose mg/kg x5
14 (78) 10 (42) 5 (18) 2(7) O(0) O(0) O(0) 11 (61) 5 (21) l(4) O(0) O(0) O(0) O(0)
GI tumors (%)
0 0 0 0 0 0 0 0 0 0 0 0 0 0
Stomach
0 1 0 0 0 0 0 0 0 0 0 0 0 0
Small intestine
TUMOR INCIDENCEa IN F 344 RATS TREATED WITH DMH
TABLE 1
Bb 5 2 0 0 0 11 5 1 0 0 0 0
.14
Colon
8 3 1 0 0 0 0 2 0 0 1 0 0 0
Zymbal’s gland
0 0 0 0 0 0 0 2 0 0 0 0 0 0
Kidney
1 2 1 3 1 0 1 4 10 3 1 0 1 2
Foci cell alteration
Liver
0 0 1 0 0 0 0 0 1 1 0 0 0 0
Neoplastic nodule 0 0 0 0 0 0 0 0 0 1 0 0 0 0
‘_
44 50 52 55 59 61 64 44 49 53 56 60 62 64
Carcinoma Weeks at sac.
274
persistant d&rease in the rate of body weight gain after 4 weeks exposure to the highest dose (30 mg/kg) of DMH in males and after 3 weeks in females treated with MAMAc. Several months later, as neoplasia became evident with associated weight loss, males and females exposed to both compounds at the 30 mg/kg dose showed less weight gain. There was a dose response with regard to the total number of tumor bearing rats, organ location and latent period of the tumors induced by both DMH and MAMAc (Table 1 and 2). The first tumors were observed at 22 weeks in the colon of 2 male rats which died after being exposed to DMH (one each 30 and 15 mg/kg). The first tumors (colon) in rats exposed to MAMAc (30 mg/kg) (1 male and 1 female) were found at 26 weeks. Several other animals in the higher dose groups of both chemicals died or were killed in a moribund state because of neoplasia prior to the termination of the study. Tumors were observed most commonly in the gastrointestinal tract, Zymbal’s gland, kidney, liver and mammary gland and occasionally up to 4 different organs showed neoplasia within the same animal (Tables 1 and 2). In the lower dose groups the tumors were confined almost entirely to the colon. Stomach All tumors observed in the stomach were in rats exposed to MAMAc (Table 2). They were confined to the non-glandular portion (forestomach) and varied from small squamous papillomas (9 rats) to invasive squamous cell carcinomas (6 rats). No metastases were observed. Small intestine Tumors of the small intestine were rare, but were found with both DMH and MAMAc (30 and 15 mg/kg, male only). All tumors were epithelial in origin and most appeared to arise in the middle portion of the small intestine in or near a lymphoid (Peyer’s) patch. Large intestine Tumors of the large intestine were more frequently observed in males than females at the same dose and DMH caused tumors at a lower dose than MAMAc (Tables 1 and 2). The location was the same with both compounds with a majority located in the proximal or distal portions of the colon. Only one neoplasm was found in the cecum and none in the anus. Most of the rats had fewer than 3 tumors in the colon but occasional animals had up to 10. All tumors of the colon were of an epithelial type and varied from carcinoma in situ to small well differentiated adenomas, polypoid carcinoma, tubular adenocarcinoma and mutinous (signet ring) carcinoma. The mutinous type often metastasized regardless of the dose of the carcinogen or sex of the animal; most frequently to the regional lymph nodes and on the serosal surface of adjacent viscera. Again, many of the smaller tumors appeared to arise in the area of a lymphoid patch, especially in the proximal colon. With one possible exception (male rat-MAMAc) the multistage model was found to provide an excellent fit to the observed tumor rates (Figs. 1 and 2).
M M M M M M M F F F F F F F
30 15 7.5 4 1 0.2 0 30 15 7.5 4 1 0.2 0
18 24 25 27 26 27 30 18 24 27 27 27 27 30
N
16 12 4 0 0 1 1 11 6 1 2 0 0 6
Total rats with tumors
aNumber of rata with tumors.
Sex
Dose mg/kg x5
12 (67) 11 (46) 2( S) 0( 0) 0( 0) 0( 0) 0( 0) 6 (44) 5 (21) 1( 4) 0( 0) 0( 0) 0( 0) 0( 0)
GI tumors
5 3 0 0 0 0 0 4 3 0 0 0 0 0
Stomach
2 0 0 0 0 0 0 0 0 0 0 0 0 0
Small intestine
10 10 2 0 0 0 0 6 2 1 0 0 0 0
Colon
TUMOR INCIDENCEa IN F 344 RATS TREATED WITH MAMAc
TABLE 2
6 0 0 0 0 0 0 0 0 0 1 0 0 0
Zymbal’s gland
6 0 0 1 0 0 0
0 0 0 0 0 0 0
Kidney
6 16 5 4 1 1 1 5 10 7 1 0 0 2
Foci cell alteration
Liver
0 1 0 0 0 0 0 2 0 0 1 0 0 0
Neoplastic nodule
0 0 0 0 0 0 0 0 0 0 0 0 0 0
46 52 56 59 62 64 64 46 53 55 54 61 63 64
Carcinoma Weeks at sac.
ul
Y
276 80-
70-
+ Male DMH O--Female DMH
0
5
IO
Fig. 1. Dose response
15 Dose
20 25 mg /kg x 5
curves for colon
30
tumors of male and female
rats exposed
to DMH
609 ? 8 50E 0 ‘Z 402
O-Male MAM o---Female MAM
Dose- mg/ kg x 5 Fig. 2. Dose response MAMAc.
curves for colon
tumors
of male and female
rats exposed
to
277
Zymbal gland
Tumors of the Zymbal gland were usually unilateral and often presented as a fungating or caseous mass at the base of the ear. They were more common in males than females. Histologically, they all resembled a squamous cell carcinoma with various degrees of anaplasia. Necrosis and inflammatory response often obscurred the tumor. No evidence of metastasis was observed. Preputial gland
The preputial gland tumors were observed in MAMAc (one 15 mg and one 7.5 mg) treated rats only. They appeared as small, hard nodules which histologically resembled the Zymbal gland tumors. Kidney
Tumors of the kidney were all found in female rats and more so with MAM than DMH. They were unilateral and varied from small cortical foci to highly invasive tumors composed of spindle shaped cells which involved most of the organ. No evidence of metastasis was found. Liver
Hepatocellular carcinoma and neoplastic nodules [ 151 were encountered rarely, and the former only in the rats exposed to MAMAc. However, foci of cellular alteration were noted at the higher doses of both compounds, but more so with MAMAc (Tables 1 and 2). They were composed of basophilic or eosinophilic or mixed-cell types [ 151. Mammary
gland
Mammary gland tumors were encountered with low frequency at the higher doses of MAMAc, in both male (one 7.5 mg) and female (one 30 mg and one 15 mg) animals. One similar tumor was observed in a female control. Microscopically, all were similar and were classified as cystic, fibroadenomas. DISCUSSION
The objective of this study was to obtain a dose of either DMH or MAMAc which would produce a relatively low incidence of neoplasia of the large intestine while at the same time producing little toxicity or neoplasia at other sites in the body. While a great deal of information is available on the morphology [9] of tumors produced by these compounds none could be found which showed a dose response relationship. However, subsequent to the initiation of this study a dose response experiment using a single subcutaneous (s.c.) dose of AOM in male Fischer rats has been reported [16]. The author found that a single S.C.injection of 0.8 mg/kg of AOM produced an incidence of 20% intestinal neoplasia, all of which was restricted to the large intestine with no evidence of neoplasia at other sites. Another recent study [lo] demonstrated that dosing sequence affects the target site. Daily S.C.injections of DMH in rats produced tumors in the large intestine only, while
278
larger doses given once to twice weekly stimulated tumors in various segments of the gastrointestinal tract (total dose was approximately equal). Both DMH and MAMAc showed a dose response relationship with a greater dose being required to produce intestinal tumors in female than male rats. In addition, DMH produced more intestinal tumors at lower doses than MAMAc. Importantly, as the dose of either compound was reduced, the incidence of tumors at other sites (Zymbal’s gland, kidney, liver) also decreased. ACKNOWLEDGEMENT
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