(Plates XLI-XLIII) Kimio YASUHIRA (Department of Pathology, Chest Disease Research Institute, Kyoto University*2)

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1 [GANN, 59, ; June, 1968] UDC [ ]: INDUCTION OF MALIGNANT LYMPHOMA AND OTHER TUMORS DURING EXPERIMENTS WITH ANTHRALIN PAINTING OF MICE*1 (Plates XLI-XLIII) Kimio YASUHIRA (Department of Pathology, Chest Disease Research Institute, Kyoto University*2) Synopsis During experiments with skin papilloma production in mice, many animals died unexpectedly due to lymphomas or other tumors. Malignant lymphomas appeared in ICR mice treated with daily paintings of Anthralin (1,8,9-trihydroxyanthracene) in acetone. The incidence of such tumors increased by intraperitoneal injection(s) of urethan prior to the first painting; over one-half of these animals developed tumors within 28 weeks. Many superficial and some deep lymph nodes were affected, and the thymus, liver, spleen, and kidneys were infiltrated by tumor cells, extensively in some cases. Histological examination showed marked proliferation of stem-cells and/or lymphoblasts. Thymomas and lung adenomas were also induced. Mammary carcinoma appearing frequently in CFW mice was thought to be spontaneous and not induced. INTRODUCTION In the previous study,12) Anthralin was found to be carcinogenic to mouse skin, as this was able to induce papillomas at the site of painting, with or without pretreatment with urethan. Therefore, this chemical may also induce tumors other than skin papillomas. In fact, a large number of mice painted with Anthralin died of malignant lymphomas. Since the pioneer work of Bungeler3) and of Lignac,9) it has been shown that murine leukemias can be induced by the application of hydrocarbons or other carcinogens. Tumor induction depends on the strain susceptibility to the chemicals applied. Most of the induced tumors have been lymphomatous and located in the thymus and/or lymph nodes. Anthralin is a hydrocarbon which has been used clinically in the treatment of psoriasis, and its irritative action on the skin has been noted. During the experiments for testing the cocarcinogenic activity of this chemical by painting on mice, enlarged lymph nodes appeared and progressed fatally. These malignant lymphomas were seen mainly in ICR and rarely in CFW and dd strains of mice. The thymomas and lung adenomatosis that appeared also in this experiment may have been due to urethan used as the initiator in this experiment in place of 7, 12-dimethylbenz[a]- anthracene (DMBA) used in Bock's experiments.1) The histological findings and possible histogenesis of these tumors are discussed in this paper. *1 This study was presented at the 25th Annual Meeting of the Japanese Cancer Association in Osaka, 1966.

2 K. YASUHIRA MATERIALS AND METHODS Materials and methods are the same as described in detail in the preceding paper. 12) Anthralin or croton oil in acetone was painted on the back of ICR or CFW young adult mice five times a week, with or without previous intraperitoneal injection of urethan. The number of tumors induced was tabulated every week and all the mice were sacrificed for histological examination at the end of the experiment. These experiments were carried out at Dr. Bock's laboratory in Roswell Park Memorial Institute in One additional experiment (Expt. 4) using dd mice was done at the author's laboratory in Kyoto. This strain of mice was propagated at, and was supplied by, the Animal Center of this University. The mice were kept in a room controlled at a in the preceding experiments and many of them died of pulmonary infection. All the mice were observed weekly and at autopsy, and histological examinations were performed as in the former experiments. RESULTS Papillomas occurring in the painted skin have been described elsewhere. During the first experiment, one mouse in Group 3 and two in Group 5 died in the 14th week of the experiment. Marked enlargement of the liver and spleen was found at autopsy. Two others in Group 5 died of thymoma in the 15th week. Mice with enlarged axillary Fig. 1. Malignant lymphomas in mice pretreated with urethan and painted with Anthralin No. 178 (left) died 17 weeks and No. 182 (right) died 18 weeks after the first painting. Shaded organs were infiltrated by tumor cells. Figures refer to weight in mg. 196 GANN

3 INDUCTION OF MALIGNANT LYMPHOMA WITH ANTHRALIN PAINTING and inguinal lymph nodes appeared one after another in the following weeks. Retroperitoneal lymph nodes were also enlarged, causing paralysis of the legs. Among the surviving mice, superficial lymph nodes were palpable in 12 of Group 5 in the 16th week and in 6 more in the 17th week. By the end of the experiment (27 weeks), a total of 18 mice had died with lymph nodes over 15mm in diameter, plus thymoma, hepatosplenomegaly, anemia, or generalized edema (Fig. 1). Three others were found at final autopsy to have malignant lymphomas. It should be pointed out that the palpable lymph nodes regressed in 4 of the mice in this group. In Group 3 of the first experiment, 4 mice had lymphomas in the 16th week, 10 in the 20th week, and 18 in the 23rd week. The lymph nodes were not so large in this group as in group 5 and there were only 4 deaths from lymphoma by the end of the experiment. Two others were found to have malignant lymphoma at the final autopsy. Three mice in Group 1 died of thymoma. Fig. 2 shows the cumulative number of deaths in these three groups of the first experiment, suggesting the simultaneous occurrence of first tumors in all groups. Fig. 2. Cumulative number of death due to malignant lymphoma Multiple foci of pulmonary tumors were seen at autopsy in the lungs of mice pretreated with urethan. Many of them were localized in the marginal portions of the lung. The appearance of these foci was quite similar to the description of the lung adenomatosis induced by urethan or other carcinogens. The number of mice bearing these tumors appears in Table I. Some animals died of renal damage (glomerulitis and cystic degeneration) causing edema and anemia, and were omitted from the table. Table I also indicates the occurrence of tumors in Expts. 2, 3, and 4. The data show that production of malignant lymphomas in Expt. 2 parallels that in Expt. 1. However, lymphomas appeared in Expts. 3 and 4 only exceptionally. Mammary tumors developed in all the groups of mice in Expt. 3, suggesting that the treatments accelerated production of this tumor, which probably occurs spontaneously in CFW mice. 59(3)

4 K. YASUHIRA Table I. Histological Findings at Autopsy Histological Findings Malignant Lymphoma: Enlarged lymph nodes appeared in the axillary, inguinal, submaxillary, mesenteric, and retroperitoneal areas. Histologically these nodes were extensively affected with proliferating tumor cells (Photo 1). The thymus, liver, spleen, kidneys, and subcutaneous tissue at the site of painting were also involved (Photos 2, 3, and 4). The protoplasm of the tumor cells was thin and almost invisible in sections stained by Hematoxylin and Eosin. Their nuclei were oval and somewhat irregular in shape and size. Chromatin was dense in the peripheral portion of the nucleus, and one or two distinct nucleoli were visible. The appearance was similar to that of lymphoblasts (Photo 5). The characteristic appearance of the cells was more obvious in metastatic foci. They appeared to be more immature, with larger nuclei and more basophilic protoplasm than in lymphoblasts. They resembled closely the so-called stem cells (Photo 6). Furthermore, myeloid cells in various stages of development were seen in other parts of the metastatic foci (Photo 7). This morphological diversity of the proliferating cells was quite marked in the early stage of tumor development. For example, the thymomas occurring at the earliest stage of tumor production consisted mainly of mature and immature plasma cells (Photo 8), and infiltrating cells in mice dying from hepatosplenomegaly were predominantly myelocytic. Later, proliferation of lymphoblastic cells predominated. In tumors occurring at a later stage, infiltrating cells were uniformally lymphoblasts or stem cells, and they tended to metastasize heavily to the kidneys, liver, lungs, and remote lymphoid tissues. It was of interest that tumor 198 GANN

5 INDUCTION OF MALIGNANT LYMPHOMA WITH ANTHRALIN PAINTING cells also infiltrated the subcutaneous tissue at the site of painting. This was noted in mice treated only with Anthralin. Lung Adenomatosis: Multiple foci in the lung were histologically adenoma or adenocarcinoma (Photo 9). They ranged in appearance from only Shimkin's hyperplastic focus. to typical adenocarcinoma. Mammary Tumor: Mammary tumors in CFW mice appeared to be carcinoma, but with somewhat different histological findings from those occurring spontaneously in C3H and from those induced chemically. Some of them consisted of small cells tending to form numerous small acini (Photo 10), the appearance resembling the carcinoma in C3H. The others were hemangioma-type, containing blood or blood cells in their central lumina. The lumina were small, surrounded by multiple layers of tumor cells in some cases (Photo 11 and 12), and were cystic with thin-walled tumor cells in other cases. All of these tumor cells were characteristic in their tendency to form small acini. DISCUSSION Murine leukemias have been induced by injections of indole,3) benzene,9) dibenz [a, h]- athracene (DBA),5) and other chemical carcinogens. Painting with 3-methylcholanthrene,6,11) DBA,7) or tar2) has also induced leukemias in mice, many of which were lymphomatosis. In these experiments, it has become clear that the chemical induction of leukemias depends on strain susceptibility as in spontaneously occurring leukemias. In the experiments described in the present paper, it was found that Anthralin painting can induce malignant lymphoma in ICR, but not in CFW or dd strain of mice. The majority of malignant lymphomas in mice have been thought to originate in the thymus, since thymectomy prolongs the life span of mice with spontaneous or induced leukemias even that of high leukemia strains.4,8,10) Another possibility is that any lymphoid tissue may become a target organ of tumor induction, because leukemias can also be induced in thymectomized animals. In the present experiment three types of leukemia were noted; thymoma, hepatosplenomegaly type of leukemia, and generalized lymphoma. The former two arose in mice treated previously with urethan and were predominantly composed of plasma cells or myeloid cells. The third occurred in mice painted with Anthralin and consisted of proliferating lymphoid cells. It was also noted that malignant lymphoid cells occurred concomitantly with plasma cell or myeloid cell proliferation and became predominant in the advanced stage of tumor progression. These observations do not completely exclude the thymic origin of lymphoma, but suggest multicentric occurrence, since enlarged lymph nodes were palpable simultaneously at various sites of subcutaneous lymph node plexuses. At any rate, it seems the lymph nodes are the target organ of the tumorigenic action of Anthralin in mice. It should also be pointed out that the induction of malignant lymphoma by Anthralinpainting can be accelerated by pretreatment with an anesthetizing dose of urethan in mice. It was shown in previous studies that urethan can initiate skin papilloma production, although application of this drug in adult mice produces no tumor in any organ except the lung. Thus, it is possible to consider urethan to be an initiator for mature lymphoid tissue. However, the problem whether the acceleration of lymphoma produc- 59(3)

6 K. YASUHIRA tion depends on the initiating ability of urethan or on synergistic action of urethan plus Anthralin remains unanswered and requires further study. Appreciation is expressed to Dr. F. G. Bock for stimulating the author to carry out this study in his laboratory. (Received December 4, 1967) REFERENCES 1) Bock, F. G., Burn, R., J. Natl. Cancer Inst., 30, 393 (1963). 2) Brues, A. M., Marble, B. B., Am. J. Cancer, 37, 45 (1939). 3) Bungeler, W., Frankfurt. Z. Pathol., 44, 202 (1932). 4) Furth, J., J. Gerontol., 1, 46 (1946). 5) Furth, J., Furth, O. B., Am. J. Cancer, 34, 169 (1938). 6) Kirchbaum, A., Strong, L. G., Gardner, W. U., Proc. Soc. Exptl. Biol. Med., 45, 287 (1940). 7) Law, L., Lewosohn, M., ibid., 43, 143 (1940). 8) Law, L. W., Miller, J. H., J. Natl. Cancer Inst., 11, 253 (1950). 9) Lignac, G. O. E., Klin. Wochenschr., 12, 109 (1933). 10) McEndy, D. P., Boon, M. C., Furth, J., Cancer Res., 4, 337 (1944). 11) Morton, J. J., Mider, G. B., Science, 87, 327 (1938). 12) Yasuhira, K., THIS JOURNAL, 59, 187 (1968). EXPLANATION OF PLATES XLI-XLIII Photo 1. Lymph node from mouse No. 182 (Expt. 1, Group 5) bearing malignant lymphoma. Photo 5. Lymphoblastic cell proliferation in a markedly enlarged retroperitoneal lymph node Photo 6. Metastatic focus in the liver infiltrated with lymphoblasts and stem-cells from mouse Photo 7. A different part of the same specimen, infiltrated predominantly with myeloid cells. Photo 8. Plasma cell proliferation in the thymus from mouse No. 28 (Expt. 1, Group 1) which Photo 10. Mammary tumor of mouse No. 330 (Expt. 3, Group 2), consisting of small acinus- Photo 11. Mammary tumor of mouse No. 328 (Expt. 3, Group 2), consisting of cyst-forming

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