THE PRODlJCTION OF BENIGN AND MALIGNANT SKIN TUMOURS IN MICE PAINTED WITH BANTU LIVER EXTRACTS

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1 THE PRODlJCTION OF BENIGN AND MALIGNANT SKIN TUMOURS IN MICE PAINTED WITH BANTU LIVER EXTRACTS M. J. A. DES LIGNERIS, M.D. (From the South African Znstitutf for Mfdical Research, Johannesburg) One of the most striking differences in cancer localization between Europeans and several native races living in tropical and semi-tropical regions of Asia and Africa concerns the occurrence of primary liver cancer. While this form of cancer is known to be of rare occurrence among Europeans, including those living in Asia or Africa, it is, both in the Dutch East Indies and in Southern Africa, the most frequent form of cancer in native males (much less so in native females). The native races concerned are the Malays and the Bantu. The reason for the frequent occurrence of liver cancer in these races is not yet known. Some authors, remembering the inherited tendency of certain strains of mice to develop cancer in particular organs, have suggested the possibility that the localization of cancer in the liver is a racial peculiarity. Others think that there is nothing racial in this localization and that external circumstances, as exposure to such endemic diseases as schistosomiasis, malaria, and various worm infections, or the absence of vitamin A in the diet, are the responsible factors. Certain alcoholic drinks have also been suspected. The fact, shown by Berman, that, among the Bantu workers in the Witwatersrand gold mines, liver cancer occurs in a much greater proportion in those originating from Portuguese East Africa (Mozambique) than in those from the Union of South Africa-though all are of the same race-would point to an external cause. There is more malaria, more schistosomiasis, possibly also more vitamin-a deficiency and a larger consumption of alcoholic drinks in the Portuguese colony. It has been established by Gillman (personal communication) that in the South African Bantu the proportion of binucleated liver cells is five or six times greater than in Europeans. Is this to be interpreted as a manifestation of hyperplasia? Strachan, who has a very large experience of Bantu pathology, states (personal communication) that a normal Bantu liver is very seldom encountered; in most instances there is, to a varying extent, periportal smallcell infiltration, which is frequently the prelude to cirrhosis; there is usually, also, increased pigment formation, such as haemochromatosis. These facts suggest the existence of a connection between certain metabolic disturbances, possibly of a toxic origin, and the occurrence of malignant change in the Bantu liver. These disturbances are not necessarily specific for the Bantu; but they are certainly much more frequent and more pronounced than in the average European. Their nature, for the present, remains a mystery. It was thought that the examination, by various means, of liver extracts both from Bantus (with or without liver cancer) and from Europeans might 489

2 490 M. J. A. DES LIGNERIS TABLE I : Production of Tumors in Mice by Liver Extracts Number of mice I Livers extracted lrradiatioi Tuiiiors of skin FIRST SERIES Bantu, cancerous SECOND SERIES European, non-cancerous European, non-cancerous Bantu, non-cancerous Rantu, non-cancerous Bantu, cancerous l SO so In 8 mice; 2 tumors malignant (Figs. 1-5) tumors; 1 malignant 2 tumors: 1 malignant 7 tumors in 3 mice; in 2 tumors beginning malignancy 5 tumors in 4 mice: 1 malignant I 25 tumors: 7 malignant throw some light on the nature of the metabolic peculiarities of the former. Professor Kennaway, of London, to whom this idea was submitted (in January 1937), kindly agreed to have such extracts examined, fluoroscopically and otherwise, by Dr. I. Hieger. The method used for the making of liver extract is that recommended by Hieger : To each 100 grams of liver 25 grams of solid potassium (or sodium) hydroxide were added, together with 200 C.C. alcohol, free from extraneous material, such as pyridine or fuse1 oil, and previously distilled. The mixture was refluxed on a waterbath for fifteen minutes; an equal volume of water was added, and each 200 C.C. of mixture was shaken out with 100 C.C. redistilled petroleum ether. The petroleum ether was then distilled off, on a waterbath, and the residue was collected. Whenever feasible, macroscopically non-cancerous tissue was removed with scissors from the masses of cancerous tissue in the livers used. There is little doubt that minute amounts of non-cancerous tissue may have escaped removal in some cases; but for all practical purposes it may be assumed that the cancer tissue extracts were devoid of admixtures of non-cancerous epithelial cells, though they must necessarily have contained some material originating from the stroma of the cancer tissue. Some extracts were sent to London (see following paper by Hieger) ; others were used in Johannesburg for experiments on rats and mice. EXPERIMENTS Rats were injected intraperitoneally or subcutaneously with solutions of liver extract in oil; or a small incision was made in the abdomen and varying amounts of the dry extract were inserted; this was done repeatedly. Up to the moment of writing nothing has been observed beyond the formation of small granulomata with giant cells around the inserted material. Owing to the longer period necessary for the formation of tumours in rats, however, as compared to mice, the results are not yet conclusive. In view of the rather poor quality of mice available for experiments in Johannesburg, these animals were subjected only to skin painting experiments ; even so, the mortality was fairly bigh. The results, however, were definite (see Table I).

3 PRODUCTION OF BENIGN AND MALIGNANT SKIN TUMOURS 49 1 FIG. 1. EPITHELIOMA, PARTLY SPINDLE-CELL, WITH NUMEROUS MITOTIC FIGURES, INVADING ADIPOSE TISSUE, IN MOUSE PAINTED FOR EIGHT MONTHS WITH EXTRACT OF BANTV LIVER CASCER (SEE TABLE I) FIG. 2. SAME TCMOUR AS FIG. 1, SH0WIh.G INVASION OF MUSCULAR TISSUE First Series of Experiments: A total of 100 mice were given, in the interscapular region, three times a week, for a period of six months, two droplets of a 0.5 per cent solution of Bantu liver-cancer extract in petroleum ether; after six months extracts were applied twice a week, for two more months. Controls received the same amount of cholesterol in petroleum ether. At the beginning of the experiment, the hair was cut short with scissors; epilation was not done. After a time the remaining hairs fell off owing to the effect of

4 492 M. J. A. DES LIGNERIS the petroleum-ether. In the controls, which were given cholesterol dissolved in petroleumether, no tumours had developed after eight months. Of the mice treated with cancer extracts, 21 survived for eight months, and as they were at that time in bad general condition, they were killed. Papillomata were found in 8 animals, 2 being definitely cancerous, as shown in Figs These malignant tumours are seen to be typical epitheliomata, such as those forming after the application of carcinogenic substances. Second Series of Experiments: A second experimental series was begun in August Three hundred mice were divided into three groups of 100 each. The skin in the interscapular region was treated twice a week with liver extract, The first group of 100 mice received extract (dissolved in petroleumether) of cancerous Bantu livers, pooled from three cases; the second group FIG. 3. SAME TUMOUR AS FIG. 1, SHOWING PENETRATION OF MUSCLE (RIGHT) BY DOWNGROWTII OF SPINDLE-CELL EPITHELIOMA (LEFT) received extract of non-cancerous Bantu livers, pooled from four cases; and the third, extract of non-cancerous European livers, pooled from three cases. Clinical and pathological details as to the cause of death in the non-cancer cases were not available. Of each group of 100 mice, were irradiated with roentgen rays at the beginning of the period of liver-extract application, at the suggestion of Professor Kennaway. The method used was that of Mayneord and Parsons. The preliminary results at the time of writing (April 12, 1940) are as follows : mice, irradiated, treated with non-cancerous European liver extract : 32 survivors; no tumour. mice, not irradiated, treated with non-cancerous European liver extract : 40 survivors; no tumour. mice, irradiated, treated with non-cancerous Bantu liver extract: 32 survivors; 3 papillomata, one of which is malignant (histologic examination),

5 PRODUCTION OF BENIGN AND MALIGNANT SKIN TUMOURS 493 mice, not irradiated, treated with non-cancerous Bantu liver extract: 41 survivors; 2 papillomata, one of which is malignant (histologic examination), mice, irradiated, treated with Bantu cancerous liver extract: 29 survivors; 7 papillomata in 3 mice. Histologic examination shows beginning malignancy in 2 papillomata. mice, not irradiated, treated with extract of cancerous Bantu livers: 42 survivors; 5 papillomata in 4 mice. One of the papillomata is shown by microscopic examination to be malignant. The results of this second series of experiments will become complete only in the course of the next six months. The preliminary observations are given here, however, since they support and extend the results of the first series, FIG. 4. ANOTHER MOUSE TUMOUR, PRODUCED BY EXTRACT OF BANTU LIVER CANCER: TYPICAL SQUAMOUS EPITHELIOMA and because they run parallel to those obtained by Hieger (see following paper). It cannot be said that irradiation has had, so far, a definite effect on tumour production, but the total figures are too small to be of statistical significance. DISCUSSION At about the time (January 1937) when we submitted to Professor Kennaway our plan of examining extracts of Bantu livers for the presence of products of metabolic disturbances 'which might bear some relationship to the puzzling liability of Bantu and other natives of tropical or semi-tropical countries to cancerous disease of the liver, experiments were published (these publications only came to our knowledge much later) by Shabad of Leningrad, who claimed to have obtained two sarcomata and one carcinoma in mice by injecting them with benzene extracts from the liver of a patient with stomach cancer and multiple metastases, though the liver itself contained no metastatic nod-

6 494 M. J. A. DES LIGNERIS ules. Shabad interpreted these results as the first direct experimental demonstration of the existence of endogenic carcinogenic substances in the human organism outside of the cancer itself. Some months later, he recorded additional examples of tumour production in the same series of experiments.l In 1938 Neufach and Shabad published results obtained by injecting mice with benzene extracts of bile from patients with malignant tumors. These experiments followed the work of Peacock and Chalmers, who showed spectrographically that tumour-producing hydrocarbons introduced into the organism are excreted in the bile. The tumours in Neufach and Shabad s mice did not, however, arise at the site of injection, but developed in distant organs. Some were benign, some malignant, and the question arises whether they were not perhaps of spontaneous origin, though the authors state that some of these FIG. 5. SAME TUMOUR AS FIG. 4, SEOWIh G PENETRATION OF MUSCLE OF EPITHELIOMA (RIGHT) (LEFT) BY DOWNCROWTIi tumours had never before been observed to occur spontaneously in the strains employed for their experiments. In a recent article Shabad expresses himself in favour of the theory that carcinogenic compounds lead to tumour formation by the way of a general action on the organism, and that spontaneous tumor formation is similarly produced by the action of endogenous carcinogenic substances. Both exogenous and endogenous carcinogenic substances may exhibit a pronounced organotropicity, as was demonstrated also by Japanese investigators who recorded the production of liver tumours in rats receiving ortho-amino-azotoluol with their food, Our experiments show that not only extracts of cancerous livers, but even those of non-cancerous Bantu livers may have the property of producing be- For a summary of Shabad s work to date see preceding paper in this issue, by Kleinenberg, Neufach, and Shabad.-ED.

7 PRODUCTION OF BENIGN AND MALIGNANT SKIN TUMOURS 495 nign and malignant skin tumours in mice. Extracts of non-cancerous European livers have not as yet been found to exhibit this property. It would be premature to speculate on the nature of the substance or agent responsible for the cancer-producing power of cancerous and non-cancerous Bantu livers. Without in any way excluding the possible activity of a virus brought to life through the carcinogenic substance present in the extract, it seems reasonable to suggest that there has been in the livers, the extract of which has shown cancer-producing properties, a metabolic disturbance of some kind, and that the result of this disturbance is the endogenic production of a substance with cancer-producing effects, both on human liver cells and on mouse skin cells. Both the treatment received by the liver tissue during extraction, and the fact that the carcinogenic effect was exerted on cells as different genetically as mouse skin cells, are strongly opposed to the possibility that the extract contained a virus. If a virus plays a rble, it can be only an indirect one, and the tracing of the origin of the cancer-producing substance and the discovery of its nature would still be of more importance from the point of view of practical carcinogenesis than the establishment of guilt on the part of a virus. S u M MARY Benign and malignant skin tumours (papillomata and epitheliomata) were produced in mice painted with extracts of cancerous and non-cancerous Bantu livers. The possible significance of these results is discussed. BIBLIOGRAPHY BERMAN, CH.: Proc. Transvaal Mine Medical Officers Assoc., vol. 15, p. 83, June BONNE, C.: Nederl. tijdschr. v. geneesk. 76: 438, CHALMERS, J. G.: Biochem. J. 32: 271, DES LIGNERIS, M. J. A. : 2d International Cancer Congress, Brussels, 1936: Communications, vol. 11, 1937, pp. 593, 602. MAYNEORD, W. V., AND PARSONS, L. D.: J. Path. & Bact. 45: 35, NEUFACH, S. A., AND SHABAD, L. M.: Bull. biol. et mcd. expcr. 6: 259, 1938 (Russ.). PEACOCK, P. R.: Brit. J. Exper. Path. 17: 164, SHABAD, L.: Bull. biol. et mcd. expcr. 3: 252, 1937; 5: 3, Acta med. URSS 2: 34, 1939 (Russ.). STRACHAN, A. S.: J. Path. & Bact. 39: 209, 1934.