[GANN, 53, ; September, 1962]

Transcription

1 [GANN, 53, ; September, 1962] COMBINED EFFECT OF CARCINOGENS WITH DIFFERENT ACTIONS III. DEVELOPMENT OF SKIN CANCERS IN THE RAT BY FEEDING 4-DIMETHYLAMINOSTILBENE FOLLOWING INITIAL PAINTING OF 20-METHYLCHOLANTHRENE (Plates XXXIII and XXXIV) Shigeyoshi ODASHIMA (The Medical Institute of Sasaki Foundation*) SYNOPSIS 20-Methylcholanthrene (MC) was painted for 1-5 months on male Wistar rats, followed by intervals of up to 12 months without any treatment, and then fed with 4-dimethylaminostilbene (DAS) for 5 months. Skin tumor was produced in various groups and it was especially interesting that skin cancer developed in the rats which received MC-painting only 8 times during the first 1 month, followed by an interval of 11 months without any treatment, and then DAS-feeding for the next 5 months. Many past reports described the incidence of skin cancer in mice by application of carcinogenic hydrocarbons1-4,9) and even painting only a small amount of 20- methylcholanthrene (MC) is generally accepted as a useful method to produce skin cancers in mice. Concerning the development of skin cancer in rats using such substances, only a few experiments have been repeorted, such as by Morris et al.,5) Nakano,6) and Watson,10) and it has been demonstrated that only prolonged application of the substances can develop skin cancer but with low incidence. The present experiment was carried out by feeding of 4-dimethylaminostilbene (DAS) for 5 months following initial painting of MC for various periods, with an interval of 7-11 months between these treatments, and the incidence of skin tumor was investigated. MATERIALS AND METHODS Animals Seven-week-old male Wistar rats, 68 heads in total, were employed. They were housed 2 to a cage and given diet and water ad libitum. Basic and DAS Diets A semi-synthetic cube diet of Oriental Co. was used for the basic diet. DAS-diet was made by the addition of DAS in proportion of 0.005% to the basic diet. MC-Painting 0.3% MC-solution in acetone was dropped, 2 times a week, to the

2 previously shaved central region of the back. The volume of each drop was about 0.05ml. Experimental Group and Procedures The animals were divided into 5 groups (Groups I-V) and each group received the MC-painting for 1-5 months. All the surviving animals were kept on a basic diet until the end of the 12th experimental month and this was followed by DAS-feeding for the next 5 months. Histological Examination The skin tumor, ear duct, liver, spleen, lungs, thymus, and mesenterial and mediastinal lymph nodes were examined. They were fixed in Bouin's solution and made into paraffin sections. The stainnig methods employed were Hematoxylin-Eosin staining, Azan-staining, and PAS-staining after glycogen digestion. Fig. 1. Schedule of animal experiment and results (The figures in the columns show number of animals employed and survived, and those outside are the valid number of animals) RESULTS At the begining of DAS-feeding, in the 12th experimental month, 17/20, 12/12, 11/12, 9/12, and 12/12 were surviving in Groups I-V, respectively. During the following 5 months of DAS-feeding, however, many rats died because of pneumonia, acute or subacute liver necrosis, intestinal bleeding, or of accidents. Thus, at the end of the DAS-feeding, only 4/17, 6/12, 5/11, 5/9, and 4/12, survived in each of the groups I-V, respectively. The first tumor animal, the liver tumor animal, was detected on the 500th experimental day. Thereafter, all the survivors were examined by inspection and palpation, for the presence of tumors once every 10 days. When the tumors were detected, the animals were kept under observation for the following days and were then sacrificed under ether narcosis for an autopsy. The number of tumor rats increased gradually and all the rats except one developed the tumor by the end 270

3 of 22nd experimental month. At that time, all the survivors were sacreficed and served for autopsy. Incidence of Tumor The number of rats in which the skin tumor developed were 4/4, 6/6, 3/5, 1/5, and 3/4 in Groups I- V, respectively. In addition, liver cancer developed in 0/4, 0/6, 1/5, 2/5, and 1/4, and ear-duct cancer developed in 0/4, 0/6, 1/5, 1/5, and 0/4 in Groups I-V, respectively. These different tumors were found independently in separate animals and finally, rat was found without any tumor. incidence of tumors each experimental month is summarized in Table I. only one The in each group in Pathology of Tumors One to eight macroscopically separated tumors in each rat were found not only in the central region but also at the peripheral region of the painted site (Photo 1). Localisation of the skin tumors in each rat was not so different in each group. The size of the tumor was also similar in all the groups and the largest one was about the tip of a thumb (Photo 2). The average number of the developed tumors in each rat in each group was about proportional to the period of MC-painting and it was 4.8, 4.8, 4.3, 4, and 3.1 in Groups I-V, respectively. Histological classification of the induced skin tumors were made on 1-4 Table II. Histological Picture of the Induced Skin Tumors large tumors in each animal and its A result is summarized in Table II. As shown in this Table, out of 45 tumor nodules examined, 15 were keratoacanthoma (Photo 3), 20 were basal cell carcinoma (Photo 4), 8 were squamous cell carcinoma (Photo 5), and the remaining 2 were spindle-cell carcinoma (Photo 6). The number of animals in which the squamous cell carcinoma or. spindle cell carcinoma developed were 2/4, 4/6, 1/3, 0/1, and 1/3 in Groups I-V, respectively. Keratoacanthoma B Basal cell carcinoma C Squamous cell carcinoma D Spindle cell carcinoma Table I. Incidence of Skin Tumors a) Liver cancr b) Ear-duct cancer Besides the tumors mentioned above, an adenoma of the sebaceous gland was detected in 2 cases (Photo 7). 271

4 Histological examinations revealed a most interesting fact that these different kinds of skin tumors were found in the one and the same animal in 14 out of 17 tumor rats and that, in some cases, only a small amount of connective tissue was found between the two tumors (Photo 8). Although the incidence of metastasis to the regional lymph node was not detected at all, the invasion of tumor clusters into the lymph vessels was demonstrated in 2 cases. These were all cases of squamous cell carcinoma. On the other hand, besides skin tumors, 4 cases of liver cancer and 2 cases of earduct cancer were found. Histological picture of the former was a typical hepatoma and that of the latter was squamous cell carcinoma. Evident histological changes were not found in other organs excepting chronic pneumonia in these tumor animals. DISCUSSION Concerning the development of rat skin cancer, only a few experiment has been reported.5,6,10) All of these reports described the low incidence of the tumor and showed natural resistance of rat skin epithelium to carcinogenic hydrocarbons. In the present experiment, skin cancer developed in a high proportian by feeding of DAS for 5 months following initial painting of MC with long intervals between these treatments. The most interesting observations made in the present series of experiments were the following points. The development of skin tumors, 1 case of squamous cell carcinoma, 1 case of basal cell carcinoma, and 3 cases of keratoacanthoma, was found in the rats which received MC-painting only 8 times during the first 1 month, no treatment for 11 months, and DAS-feeding given for the next 5 months. Ear-duct cancer is developed in the rat8) by continuous feeding of only DAS. This may be accepted as the result of essential natural affinity or sensitivity of the rat ear-duct epithelium to DAS metabolites. The results obtained in the present experiments, on the contrary, will be interpreted as follows. The cells which showed higher sensitivity to DAS than the ear-duct epithelium of a normal rat were produced by only 8 times of MC-painting, these sensitized cells kept their characteristics for the following 11 months, and then reacted with the DAS administered. This interpretation will be accepted with only one exception of the following possibility that an unknown age factors acted on the painted epithelium and developed the skin tumor spontaneously. This possibility cannot be entirely excluded because no one had kept rats under observation for so long a period as 21 months after only 1 months of MC-painting, but the possibility seems to be very small. In the previous experiments,7,8) transplantability as well as the incidence of lung metastasis of azo dye-induced liver cancer was examined and the results showed that they were both proportional to the period of DAB-feeding; malignancy of the induc- 272

5 ed liver cancer seemed to depend on the dose of carcinogens given. Skin tumors may be an interesting material to analyse such a problem because histologically different types of tumors could be developed, but, in the present experiments, such correlations were not studied fully, because only 1-6 cases of tumor rats could be studied on the development of skin tumors. SUMMARY The development of skin tumors was studied on the rats which received MCpainting for 1-5 months, followed by an interval of 7-11 months without any treatment, and then feeding of DAS for 5 months. Results obtained were as follows: 1) In all the animals given MC-painting for 4-5 months, skin tumor alone was produced. 2) Skin cancer development was found even in the rats which received MC-painting only 8 times during the first 1 month, followed by an interval of 11 months without treatment, and then DAS-feeding for the next 5 months. Based on these results, the carcinogenic actions of MC and DAS were investigated and discussed in relation to the development of skin cancer in rats. This work was carried out under the direction of Prof. Tomizo Yoshida, University of Tokyo and Director of this Institute, and Prof. Morizo Ishidate, Director of Tokyo Biochemical Research Institute, to whom the author is greatly indebted for invaluable suggestions and criticism during the course of this investigation. REFERENCES 1) Barry, G., et al., Proc. Roy. Soc. (London), (B), 117, 318 (1935). 2) Bechmann, W.E., et al,, ibid. 123, 343 (1937). 3) Cook, J.W., et al., ibid. 111, 455 (1932). 4) Iball, J., Am. J. Cancer, 35, 188 (1939). 5) Morris, H.P., et al., Cancer Research, 7, 730 (1947). 6) Nakano, K., Osaka Igaku Zasshi, 36, 483 (1937). 7) Odashima, S., THIS JOURNAL, 50, 321 (1959). 8) Idem, ibid., 53, 247, 259 (1962). 9) Roe, F.J.C., Brit. J. Cancer, 10, 61 (1956). 10) Watson, A.P., J. Pathol. Bacteriol., 34, 301 (1931). 273

6 EXPLANATION PLATES (XXXIII and XXXIV) Photo 1. Multiple skin tumors developed in the rat which received DAS-feeding for 5 months after an interval of 7 months from initial MC-painting for 5 months. Sacrificed after 610 experimental days. Photo 2. The largest skin tumor developed in the rat which received DAS-feeding for 5 months after an interval of 9 months from initial MC-painting for 3 months. Sacrificed after 645 experimental days. Photo 3. Adenoacanthoma of the rat shown in Photo 1. In the same animal squamous cell carcinoma was also found. Photo 4. Basal cell carcinoma. Photo 5. Typical squamous cell carcinoma. Photo 6. Spindle-cell carcinoma. Photo 7. Benign sebaceous adenoma. Photo 8. Two kinds of skin tumors separated by a thin strand of connective tissue. Benign sebaceous adenoma (right) and basal cell carcinoma (left). 274

7 GANN, Vol. 53 PLATE XXXIII

8 GANN, Vol. 53 PLATE XXXIV