NOTE. [GANN, 65, ; August, 1974]

Transcription

1 NOTE [GANN, 65, ; August, 1974] COMBINED EFFECT OF VARIOUS SURFACTANTS ON GASTRIC CARCINO- GENESIS IN RATS TREATED WITH N-METHYL-N'-NITRO-N-NITROSOGUANI- DINE*1 Shoji FUKUSHIMA, Masae TATEMATSU, and Michihito TAKAHASHI First Department of Pathology, Nagoya City University Medical School*2 Experimental gastric carcinogenesis has made a great stride in the induction of adenocarcinoma of the glandular stomach of rats by oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).10) We described in our previous paper11) that adenocarcinomas having marked malignant qualities developed in the glandular stomach of rats that received MNNG combined with several surfactants in the drinking water. Nonionic surfactants employed in this study, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monolaurate (Span 20), glyceryl monostearate, and sucrose monopalmitate have been used as emulsifiers in foods, drugs, and cosmetics in several countries because of the harmless appraisal.1,3,5,12) Anionic surfactants, such as sodium lauryl sulfate and sodium N-lauroyl sarcosinate have been widely applied to dentifrices, cosmetics, and pharmaceutical preparation, because their toxicity is mild to human body. The present study was undertaken to evaluate the combined effect of above-mentioned surfactants on the induction of cancers of the glandular stomach in rats by MNNG. Materials and Methods Ninety-eight male Wistar rats (Nihon Rat Co., Ltd., Saitama), about 60 days old at the start of the experiment, were used. MNNG (Aldrich Chemical Company, Inc., Milwaukee, U.S.A.) was employed as a carcinogen. Tweens 20, 40, 60, and 80, Span 20, glyceryl monostearate, sucrose monopalmitate, sodium lauryl sulfate, and sodium N-lauroyl sarcosinate were used as surfactants. *1 A part of this work was reported at the 31st Annual These surfactants were purchased from Nikko Chemical Co., Ltd., Tokyo, and Dai-nippon Sugar Mfg. Co., Ltd., Tokyo. Methods of the experiment are summarized in Table I. Rats were divided into 9 experimental groups and one control. Nine or ten rats in each experimental group received MNNG solution at a concentration of 50mg/L containing various kinds of surfactant in the drinking water freely for 26 or 30 consecutive weeks. The surfactants added to MNNG were as follows: Group 1, 0.4% Tween 20; Group 2, 0.4% Tween 40; Group 3, 0.4% Tween 60; Group 4, 0.4% Tween 80; Group 5, 0.3% Span 20 plus 1.7% ethanol; Group 6, 2% glyceryl monostearate; Group 7, 0.2% sucrose monopalmitate; Group 8, 0.25% sodium N-lauryl sulfate; Group 9, 0.5% sodium N-lauroyl sarcosinate. Ten rats in Group 10, a control group, were administrated MNNG solution alone at the same concentration for 26 consecutive weeks. After 26 or 30 weeks, all the rats were given tap water and fed commercial food (Oriental MF) throughout the experiment. The experiment was terminated at 80 weeks after the start of the drinking. Autopsy was done when the rats were found dead or killed in a poor condition, stomach and other organs were examined, and fixed in 10% Formalin solution. The tissues were routinely processed and stained with Hematoxylin and Eosin, and occasionally, special staining methods were used. Results Rats which survived 30 weeks or more after the start of the administration were considered to be effective. None of the rats receiving surfactants showed diarrhea at any time during the observation, but anemia commonly developed during the Meeting of the Japanese Cancer Association, end of the experiment. The same criteria mentioned in our previous paper11) were applied to the Nagoya, October, This study was supported in part by Grants-in-Aid for Cancer Research from diagnosis of the experimental gastric carcinoma. the Ministry of Education and from the Ministry of Health and Welfare. Cancer of the Glandular Stomach Grossly, most of *2 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya the tumors were localized in the pyloric region. As summarized in Table II, the tumors in the 65(4)

2 S. FUKUSHIMA, ET AL. Table I. Methods of the Experiment MNNG=N-Methyl-N'-nitro-N-nitrosoguanidine, GMS=glyceryl monostearate, SMP=sucrose monopalmitate, SLS=sodium lauryl sulfate, SNS=sodium, N-lauroyl sarcosinate Table II. Induction of Cancer of the Glandular Stomach in Rats by Oral Administration of MNNG Combined with Various Kinds of Surfactant Figures in parentheses show the number of sarcoma present with adenocarcinoma. glandular stomach were obtained in 9 of 10 effective rats in Group 1, in 7 of 9 in Group 2, in 7 of 9 in Group 3, in 3 of 7 in Group 4, in 9 of 10 in Group 5, in 4 of 7 in Group 6, in 9 of 9 in Group 7, in 8 of 10 in Group 8, in 5 of 8 in Group 9, and in 3 of 6 in Group 10. Microscopically, tumors were diagnosed as either carcinoma or sarcoma. According to histological characteristics, adenocarcinomas were classified further into two types, well-differentiated and undifferentiated, as mentioned previously.11) Well-differentiated adenocarcinomas were observed in 8 of 9 tumors in Group 1, in 5 of 7 in Group 2, in 5 of 7 in Group 3, in 3 of 3 in Group 4, in 8 of 9 in Group 5, in 3 of 4 in Group 6, in 9 of 9 in Group 7, in 7 of 8 in Group 8, in 4 of 5 in Group 9, and in 3 of 3 in Group 10. Many of the tumor cells had a tubular arrangement with slight structural atypism. In some part, the tumor tissue showed papillary or cystic pattern. These tumor cells were composed predominantly of cylindrical cells. Nuclei of tumor cells were relatively hyperchromatic, and situated at the base of the cytoplasm. While the muscle layer and the serosa were infiltrated by the tumor cells, no metastases were detected in any animals with these adenocarcinomas. Undifferentiated adenocarcinomas were found in 1 of 9 tumors in Group 1, in 1 of 7 in Group 2, in 2 of 7 in Group 3, in 1 of 9 in Group 5, in 1 of 4 in Group 6, and in 1 of 5 in Group 9, but there was no undifferentiated adenocarcinoma in Group 10. Tumor cells having hyperchromatic nuclei formed solid nests, or were scattered as solitary cells within fibrous stroma (Photo 1). Also, neoplastic glands having a marked structural atypism occasionally coexisted within the same tumor tissue, and the tumor cells showed a loss of polarity. In one animal in Group 6, there were a mutinous type composed of anaplastic tumor cells floating in pools of copious mucus and a scirrhous type with abundant fibrous stroma (Photos 2 and 3). These tumor cells invaded the omentum and the liver beyond the gastric serosa (Photos 4 and 6). Lymphatic invasion of tumor cells was found in 1 animal each in Groups 2, 6, and 9 (Photo 5). There were several lesions diagnosed as sarcoma either alone or mixed with carcinoma at the same time. Sarcomas were observed in 3 of 9 tumors in Group 1, in 1 of 7 in Groups 2 and 3, in 4 of GANN

3 GASTRIC CARCINOGENESIS BY MNNG in Group 5, and in 3 of 8 in Group 8. These were fibrosarcomas, hemangiosarcomas, an osteogenic sarcoma, and an undifferentiated sarcoma. One animal in Groups 2 and 8 had only a sarcoma, but others were combined with adenocarcinoma. In 2 animals with a sarcoma in Group 1, tumor cells invaded lymphatics and blood vessels in one, and the other had metastasis to the regional lymph nodes and multiple hematogenous metastases to the liver and the lung (Photos 7 and 8). No sarcomas were found in Group 10. Carcinosarcoma was not detected in any of the animals. Tumors of Other Organs Some tumors were found in the duodenum and the jejunum in every group. Histologically, they were adenocarcinomas and sarcomas. A few subcutaneous tumors were also observed. Discussion One of the purposes in this investigation was to determine whether a combination of a carcinogen and a surfactant induced the same results as in our previous experiment.11) The most interesting result obtained in the present work was a production of a few undifferentiated adenocarcinomas and several sarcomas in the glandular stomach in the animals of the experimental groups. Undifferentiated adenocarcinomas showed a high degree of anaplasia, and made an intralymphatic invasion as well as a direct spread to the omentum and the liver. Sarcomas showed a malignant quality with metastases and invasions. On the contrary, none of the rats administered MNNG alone had undifferentiated adenocarcinomas or sarcomas, and neither lymphatic invasion nor metastasis was found. It is obvious, therefore, that gastric carcinogenesis was enhanced more strikingly in the experimental groups than in the control. Surfactants seem to increase the absorption of various substances from the gastrointestinal tract.4, 6,7,9,13) In another experiment in this laboratory, we recognized that the surfactants employed in the present study promoted the absorption of Ponceau 3R from the glandular stomach and the small intestine in rats. This fact indicates that the absorption of MNNG from the glandular stomach may be promoted by the surfactants added. It has been considered that surfactants promote tumorigenesis, but there have never been reports that surfactants themselves cause gastric carcinogenesis.2,8,14) It is assumed that gastric carcinogenesis was enhanced by the action of surfactants used as a vehicle for the carcinogen. The results of the present study suggest the possibility that surfactants increase the activity of harmful chemical substances in life environment. When a surfactant is added to chemical substances, special consideration should be made for evaluation of their safety. (Received April 9, 1974) References 1) Chusid, E., Diamond, J., J. Pediat., 46, 222 (1955). 2) Dammert, K., Acta Pathol. Microbiol. Scand., Suppl., 124, 1 (1957). 3) Janowitz, H. D., Hollander, F., Marshak, R. H., Gastroenterology, 24, 510 (1953). 4) Jones, C. M., Culver, P. J., Drummery, G. D., Ryan, A. E., Ann. Intern. Med., 29, 1 (1948). 5) Krantz, J. C., Carr, C. J., Bird, J. G., Cook, S., J. Pharmacol. Exp. Therap., 93, 188 (1948). 6) Ermala, P., Setala, K., Ekwall, P., Cancer Res., 11, 753 (1951). 7) Ekwall, P., Ermala, P., Setala, K., Cancer Res., 11, 758 (1951). 8) Setala, K., Setala, H., Holsti, P., Science, 120, 1074 (1954). 9) Snyderman, S. E., Morales, S., Chung, A. W., Lewis, J. M., Messina, A., Holt, L. E., Pediatrics, 12, 158 (1953). 10) Sugimura, T., Fujimura, S., Nature, 216, 943 (1967). 11) Takahashi, M., Fukushima, S., Sato, H., Gann, 64, 211 (1973). 12) Waldstein, S. S., Schoolman, H. M., Popper, H., Am. J. Digest. Dis., 21, 181 (1954). 13) Wissler, R. W., Bethard, W. F., Barker, P., Mori, H. D., Proc. Soc. Exp. Biol. Med., 86, 170 (1954). 14) Wong, T. W., Juras, D. S., Wissler, R. W., J. Natl. Cancer Inst., 22, 363 (1959). Explanation of Plates Photo 1. Undifferentiated adenocarcinoma composed of signet-ring cells and anaplastic tumor cells with darkly stained nuclei and basophilic cytoplasms. Photo 2. Mucoid type producing large amounts of extracellular mucus. Isolated groups of tumor cells are surrounded by pools of mucus. (No. 1120, Group Photo 3. Scirrhous type with abundant fibrous Photo 4. Anaplastic tumor cells infiltrating the muscle layer. (No. 1120, Group 6). Mucicarmine stain. Photo 6. Infiltrating growth of isolated tumor cells into the liver. The arrows indicate the presence of mucus produced by the tumor cells in the hepatic sinusoids. (No. 1120, Group 6). Alcian Blue and per- H-E=Hematoxylin-Eosin stain 65(4)

4 GASTRIC CARCINOGENESIS BY MNNG 65(4)

5 S. FUKUSHIMA, ET AL. 376 GANN