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Tumor Promotion by Euphorbia Latices*

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Tumor Promotion by Euphorbia Latices* Tumor Promotion by Euphorbia Latices* F. J. C. ROE AND WiNIFRED E. H. PEIRCE (Cancer Research Department, London Hospital Medical College, London, E.1, England) SUMMARY Numerous papillomas and two malignant tumors were elicited in 101 strain mice treated once weekly with applications of acetone extracts of latices of ten species of Euphorbia after a single dose of DMBA in acetone. Of the species tested E. ti~ucalli was the most potent. Three papillomas were seen in 40 mice treated with DMBA only and three among 100 mice treated with latices only. These results indicate the presence of potent tumor-promoting agents in the latices. With one exception, there was good correlation between degree of epidermal hyperplasia and tumor-promoting effect. In the course of studies on the two-stage mecha- tion of the active principle of croton oil is an objec- nism of skin tumor production in mice and rabbits, tive of considerable importance, and it is hoped many tumor-initiating agents have been discov- that the experiments described here, in addition to ered. Several of the carcinogenic polyeyclic hydro- any intrinsic interest they may have, will lead in- carbons (e.g., 3,4-benzpyrene, 9,10-dimethyl-l,~- directly toward that goal. benzanthracene, ~0-methylcholanthrene), applied About 18 months ago a colleague, Dr. J. S. in subcarcinogenic doses, have been shown to Fawcett, of the Department of Experimental Bio- initiate tumor formation. In addition, substances chemistry, London Hospital, suggested that we which are not carcinogenic for the skin, e.g. ure- should test the latex of Euphorbia ingens for tu- than and triethylene melamine, have been shown mor-promoting activity. He had come across this to act in this way (el, e4). material during the war, while searching for alter- On the other hand, the choice of tumor-promot- natives to natural rubber, which was in short ing agent has always been much more limited. Un- supply. The latex was so irritating to human skin til recently croton oil was the only promoting that it was obviously not a suitable substitute. Be- agent of sufficient potency for reliable use in cause of this irritant effect, and because E. ingens studying the two-stage mechanism in mouse skin. belongs to the same family as Croton tiglium, from Many attempts to isolate the active principle of which croton oil is obtained, Fawcett thought it croton oil have failed (1, ~, 5, 6, 9, ~6), although might be interesting to test it for tumor-promoting highly potent fractions of it have been prepared activity. (1). Lijinsky has recently obtained crystalline In due course, a sample of the latex was ob- products (le), but these have not yet been tested tained and tested. The present paper describes ex- for biological activity. periments with the latex of E. ingens, and also Since 1953, several pure substances have been with latices from nine other species of Euphorbia. found to possess promoting activity for mouse MATERIALS AND METHODS skin; among these are iodoacetic acid (10), chlor- acetophenone (10), several surface-active agents Mice.--Male and female mice of the "101" in- (~5), and many phenolic compounds including bred strain were used for all experiments. They phenol itself (:3). Of these pure chemical substances were housed in groups of ten in zinc or galvanized phenol is the most active promoter; nevertheless, it iron cages. White wood shavings and sawdust is considerably less potent than croton oil or its were used as bedding. The mice were fed on cubed Diet 41B (obtained from Messrs. E. Dixon and resin (cf. [3] and [1]). It follows that the identifica- Sons Limited, Ware, Herts.) and water ad libitum. * The cost of this research was partly defrayed out of a All mice when 6-8 weeks old were vaccinated on Block Grant from the British Empire Cancer Campaign. the tail with sheep lymph against ectromelia. Received fo~ publication August 19, 1960. Only those showing a positive reaction were used. 338 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1961 American Association for Cancer Research. ROE AND PEIRCF~--Tumor Promotion by Euphorbia Latices 339 Technic of application to the skin.--The hair of in a few weeks, whereas none of the stock solutions the entire dorsal area from the root of the tail to showed any visible deterioration after 30 weeks at the nape of the neck was removed with electric 4~ C. in the dark. clippers from all mice before the beginning of the experiment and thereafter at ~- to 3-week inter- RESULTS vals. Experiment I: Euphorbia ingens.--Preliminary All test substances were applied to the skin from tests were carried out to see whether the latex was calibrated pipettes, care being taken to ensure that irritating to the skin of mice. Concentrations in the solution spread evenly over the entire clipped acetone above 10 per cent caused complete necro- area. sis within ~4 hours. A 10 per cent acetone extract Recording of tumors.--AU mice were examined caused patchy necrosis of the skin, which took up at fortnightly intervals for tumors of the skin and to 3 days to become manifest to the naked eye. other organs. Sick mice were killed and, together Damage to the skin was most marked when the with those found dead, examined post mortem. skin was in the resting phase of the hair cycle. His- Histology.--Specimens of skin, benign and ma- tologically, there was marked epidermal hyper- lignant skin tumors, and other tissues were exam- plasia in those areas which had not undergone ined histologically. Either Zenker's fluid or formol necrosis. Five per cent and 1 per cent acetone ex- saline was used as a fixative, and the sections were tracts caused less necrosis and more hyperplasia. stained with hematoxylin and eosin. A 0.1 per cent extract caused hyperplasia without Chemicals.--9,10-Dimethyl- 1, ~-benzanthracene necrosis. (DMBA) was obtained from L. Light and Co., and After treatment with a tumor-initiating agent, acetone (Analar grade) from British Drug Houses mice tend to develop skin tumors at the margins of Ltd. healing wounds, irrespective of how the wounds The Euphorbia latices were obtained from three are caused (13, 15, 16, 19, ~3). Therefore, in testing sources? The collection of latex from the succulent chemical substances for tumor promotion it is de- species presented little difficulty, since it flowed sirable to avoid excessive damage to the epidermis. freely from the incised stems. Collection from the With this in mind, caution was exercised in testing nonsucculent, E. wulfenii, was far more tedious: the latex of Euphorbia ingens for tumor promotion: only the cut tip of the stem could be used, and treatment was begun with a 0.1 per cent solution. each stem provided no more than 3 small drops of Test of the latex of E. ingens for tumor-promoting latex. Rubber gloves were worn to protect the activity and carcinogenicity.--Three groups of mice hands during collection. were used in the main experiment. Each group Preparation and storage of extracts.--The samples consisted of ten males and ten females. Groups I sent fr(nn South Africa were preserved by the addi- and III received a single application of 0.~ ml. 0.15 tion of a few drops of toluene immediately after per cent DMBA in acetone (300 ~g.) to the clipped collection. Shortly after receipt, 5 per cent acetone dorsal skin. Mice of Group II were clipped but not extracts were prepared from the semi-solid latices treated. Three weeks later mice of Groups I and II by grinding in a pestle and mortar, adding acetone, began to receive once-weekly applications of an shaking, and finally filtering. The resulting ex- acetone extract of E. ingens latex. The concentra- tracts were stored in amber-glass bottles at 4 ~ C. tion of the latex was held at 0.1 per cent for 3 The latex of E. ingens, and all those obtained from weeks, and then, since there was no macroscopic The Royal Botanic Gardens, were liquids. :Five per damage, increased to 0.~ per cent. After a further S cent extracts of these were prepared in the same weeks at this level, it was increased to 0.5 per cent way, except that grinding was unnecessary. More for 7 weeks. During this period the first papillomas dilute solutions were made up from the stock solu- appeared. The concentration was increased to 1 tions as required. per cent during the 14th week of secondary treat- Samples of the extracts left in the light at room ment, and to ~ per cent for the 15th and 16th temperature changed from colorless to red-brown weeks. However, the ~ per cent solution caused 1 Dr. P. R. Enslin, National Chemical Research Labora- patchy necrosis, and so the concentration was re- tories, Private Bay 191, Pretoria, South Africa, kindly supplied duced again to 1 per cent and held there from the samples of Euphorbia ingens. Dr. R. A. Dyer of the Department 17th to 3~d weeks. Except for periodic clipping, of Agricultural Technical Services, Pretoria, South Africa, sent mice of Group III received no treatment after the the samples of latex from E. tirucalli, E. grandidens, and E. initial single application of DMBA. triartgularis. The remaining latices were obtained from the Royal Botanic Gardens, Kew, Surrey, through the courtesy Papillomas began to appear on the backs of and cooperation of the Director, Dr. G. Taylor, and of Dr. mice of Group I during the 10th week of treatment F. N. Howes. with the extract. The incidence of papillomas Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1961 American Association for Cancer Research.
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