Chemical Compounds As Carcinogenic Agents Second Supplementary Report: Literature of 1938 and 1939 Biological Considerations

CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS

SECONDSUPPLEMENTARY REPORT: LITERATURE OF 1938 AND 1939

J. W. COOK AND E. L. KENNAWAY (From the Royal Cancer Hospital (Free), London, and the University of Glasgow) BIOLOGICALCONSIDERATIONS

(Continued from page 428, Jitly 1940)

II. Action of Carcinogenic Compounds in Difierent Species and Tissues A number of reports of the action of carcinogenic compounds on human tissues have appeared. Klar (601) developed a nodule on the forearm after completion of a series of experiments with 3:4-benzpyrene. He applied a solution of the hydrocarbon (0.25 per cent in benzol) to the skin of mice with a paint brush and for at least part of the period wore rubber gloves. He also conducted experiments, of which no description is given, with the powdered hydrocarbon contained in a glass vessel. Three months after the completion of the experiments a small nodule appeared on the dorsum of the left forearm, This was excised in May 1938 and described by Professor Huckel as a “ so- called benign calcifying epithelioma.” The growth extended into the subcutaneous fatty tissue; the connection with the superficial epithelium is not described nor is it evident in the two photomicrographs which illustrate the report. The author does not state his age. Gordonoff and Walthard (562) record the occurrence of a tumor in a laboratory assistant, aged forty-two, engaged in applying methylcholanthrene (0.3 per cent in benzol) to the skin of mice. The site was in the nasolabial fold, at a spot often touched by the patient when smoking. The microscopic appearance was that of a “ still well delimited stage of an incipient squamous- cell sarcoma.” Cottini and Mazzone (479) deliberately applied 3 :4-benzpyrene ( 1 per cent in benzene) to the skin, generally of the arm or thigh, of 26 patients with various cutaneous diseases, usually daily for periods up to 120 days. The changes observed were ( 1) pigmentation (“ an increase in melanin in the basal layer of the epidermis ”), which was more distinct in older people and in parts exposed to light, and (2) verrucae. The latter are described as “ an accentua- tion of the relief pattern of the skin secondary to a deepening of the sulci,” but no dimensions are given. The changes retrogressed completely within two months after cessation of the applications. In a child aged four, suffering from xeroderma pigmentosum with multiple squamous-cell cancers of the face, the skin reacted to the hydrocarbon in the ordinary way, though the reaction to ultraviolet radiation was intense. This latter effect was less in areas treated with the hydrocarbon. Application of an ointment containing 1 per cent 3:4- benzpyrene to one of the ulcerated cancers in this patient had at first a favour- 521 522 J, W. COOK AND E. L. KENNAWAY able effect, but after 30 applications no definite retardation was observed. A similar result was obtained in a man of sixty with an x-ray cancer. Oberling, SanniC, GuCrin and GuCrin (726) have published data upon carcinogenesis by 3 :4-benzpyrene in several species. (1) The hydrocarbon (1 per cent benzene) was applied twice weekly to the skin of 5 rabbits. Towards the third month 2 animals developed nodules which on biopsy showed squamous epithelioma, but in spite of continuation of the painting these tumours disappeared. At the end of one year the 3 surviving rabbits began to show malignant growths; 2 died, in the 18th and 19th months, with infiltrating epithelioma, while the third showed, in the 2 1st month, far-advanced ulcerating and vegetating epithelioma. (2) Application of the same solution to the skin of rats produced, in 3 animals which died between the nineteenth and the twenty-eighth month, epitheliomas partly of basal-cell and partly of squamous type. The production of such tumours in the skin of the rat by methylcholanthrene was previously described and illustrated by Bachmann et d. (208). (3) In guinea-pigs the same solution applied to the skin produced only traces of papilloma even when the experiment lasted more than two years. (4) Of 5 fowls similarly treated one developed an erythroblastic blood reaction, another an erythroblastic leukaemia and a tumor of the ovary, and a third, after two years, an atypical spinocellular epithelioma. (5) 3:4-Benzpyrene dissolved in lard was given with the food to 20 mice. Of 3 which lived more than six months, 2 showed epithelioma of the squamous portion of the stomach (cf. Waterman, 359, 360). Rats similarly treated developed no tumours of the alimentary tract. (6) Negative results were also obtained in rats by intrahepatic and intra- renal injections of a colloidal solution 1 in 10,000 and by injection into the kidney of a “ solution huileuse.” Of 4 rats in which a crystal of 3:4- benzpyrene was inserted into the kidney, one developed a keratinizing renal carcinoma with peritoneal metastasis, which was transmitted by grafting. (7) In an attempt to ascertain the minimum effective dose the authors found that 0.05 mg. produced sarcoma subcutaneously in one of 5 rats in a year and a half (cf. Dobrovolskaia-Zavadskaia, 501). The importance of concentration is shown by an experiment in which the same quantity of hydrocarbon was carcinogenic when injected in 0.5 C.C. of oil, but was inactive in 5.0 C.C. of the same medium. Strong and Smith (807) injected methylcholanthrene subcutaneously ( 1.O mg. in sesame oil) into male and female mice, two months old, of strains NH, HE, CBA, C57, JK, CBAN, and N. These mice had received an injection of India ink within twenty-four hours after birth. Tumours appeared in 42 of them as follows: in 16 males and 18 females, sarcoma; in 6 females of strain NH, between the 82nd and the 199th days, and in 2 females of strain JK on the 203rd and 217th days, adenocarcinoma of the mammary gland at the injection site. Females of the two closely related strains NH and JK have an extremely low incidence of spontaneous tumours, none having been met with in the former strain since its origin three years ago. The experiment was repeated without the use of India ink, and at the time of the report 2 NH mice (T CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 523 females) had developed mammary cancer. A female of this strain painted with a 1 per cent methylcholanthrene solution ( ? solvent) had also developed a mammary cancer near the site of painting. These tumours showed many areas of squamous metaplasia and were thought to have arisen from ducts. Mider and Morton (685) applied methylcholanthrene twice weekly to nine areas of the skin in turn in 65 breeding females of sub-line 212 of the dilute brown strain of mice. This sub-line has a lower incidence of mammary cancer than other members of the strain. Cancers were obtained in 13 mice between the 106th and 268th days of life (average 181st day), while 14 of 42 control breeding females developed mammary cancer between the 250th and 475th days (average 371st day). “ The number of observations does not permit any comparison of the breast tumor incidence in the two groups. The constant marked reduction in the latent period, however, is unquestionably significant.” Mammary tumours were not obtained in 75 virgin females, nor in males, treated similarly. [On the production of mammary cancers by applications to the skin, cf. Widmark, 842 (see p. 405 in the previous installment of this paper) and Maisin and Coolen, 17 1.1 Larionow (633) added 3 :4-benzpyrene or 1: 2 :5 :6-dibenzanthracene in ((oil” to the food of mice at intervals of from one to three days for from five to seven months, the whole amount given being from 9 to 19 mg. Of 60 mice, 41 survived a whole course of feeding and the first tumours appeared six months after the dosage had ceased, ie., a year from the beginning of the experiment, when 22 animals still remained alive. These mice showed: (1) papilloma of the pre-stomach in 6 instances, some multiple and one showing a transitional stage to cancer; (2) a squamous carcinoma of the pre-stomach; (3) one example each of adenoma of the sebaceous gland, mammary adenoma, and mammary adenocarcinoma; (4) adenoma of the lung in 5 instances; a single adenocarcinoma of the lung with metastases. No data are given in the French or English summaries of this paper on the incidence of tumours in control mice. Bonser and Orr (438) have described the histology of 160 tumours induced in the subcutaneous tissue of mice by methylcholanthrene (in lard or paraffin) or by 3 :4-benzpyrene or 1: 2 :5 :6-dibenzanthracene (in paraffin), which were distributed between the two sexes as follows: Male Female Sarcoma 65 60 Sarcoma and carcinoma of breast - 13 Sarcoma and squamous carcinoma 2 4 Carcinoma of breast - 9 Squamous carcinoma 1 5 Sarcoma, breast carcinoma and squamous carcinoma - 1 __ 68 92

“ The occurrence of a considerable series of mammary adenocarcinomas in female mice at the site of injection of a carcinogenic hydrocarbon and, from the histological appearances, directly attributable to the action of the hydrocarbon is a phenomenon which has not been described before. . . . As the tumours occurred close to the injected material and were as frequent in IF TABLEXIII: Obserrwltions on Eighl Highly Inbrcd Strains of Mice, Showing SyscCPribilitr lo SpMJancous Mammary and Pulmonary Tumours and Induced Sub- cvtanu~wand Pdlrrmrcrry Tumoovs. and Naiurot~mulo TranrplrmbMc Tu~rs(Andrro6nt)

CaH 95 to 100 per cent ? Medium High LOW Develop spontaneous and induced hepatomas C Less than 5 per cent 20 to 30 per cent High LOW LOW An incidence of 50 per cent of spontaneous lymphmytomas “ ” LOW LOW LOW *a M Medium to low High NI+ C57 bW Less than 1 per cent Low Low High LOW Very low incidence of spontaneous mammary tumours A 80 to 85 per cent 50 to 85 per cent Very high High Low Exdent test animals for study of sp0ntane.o~~or induced pulmonary tumours; susceptible to ulceration when injected subcutaneously D 65 to 100 per cent Low LOW LOW Good soil for propagation of well )mom transplantable mouse tumours I Less than 5 per cent Medium High High Develop spontaneous hyperplasia of the epithelium in the pyloric portion of stomach: resistant to growth of sarcoma 180 Y *’Medium ” Medium to low LOW LOW Very resistant to induction of moma by hydrocarbons but very susceptible to the growth of transplantable sarcomas CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 525 and CBA mice (which have never been observed to develop spontaneous mammary carcinoma) as in other groups, we attribute them to the presence of the carcinogenic agent.” Andervont (381,383) compared the effects in 8 strains of mice (C,H, C, C57 black, M ‘I Leaden,” A, D, I, Y) of subcutaneous injections of 1 :2:5:6- dibenzanthracene or of methylcholanthrene in lard. The results are sum- marized in a valuable table (Table XIII). In such experiments it is essential to use the same method of injection throughout; in C,H mice, for instance, a lard solution of the hydrocarbon induced tumours earlier than did colloidal suspensions or the crystalline material. The wide variance in susceptibility revealed by these strains of mice shows the necessity of using inbred animals when comparing the carcinogenic activity of different substances. Thus CsH mice might respond to weakly carcinogenic compounds which would not affect mice of strains I or Y. It is difficult to give quantitative expression to susceptibility to induced lung tumours because both the number of mice showing the tumours and the number of tumours in individual mice must be considered. C,H mice develop sarcomas at the site of injection and die before tumours are found in their lungs, while A mice develop primary pulmonary growths before sarcomas appear at the injection site. Intravenous injection thus seems to be required for comparison of susceptibility to lung tumours. This study of 8 strains of mice indicates that those which develop most spontaneous pulmonary tumours are also most susceptible to induced pulmonary tumours. This character is com- parable to the response of high mammary tumour strains to treatment with oestrogens. There is no correlation between susceptibility to spontaneous mammary tumours and to induced or grafted subcutaneous tumours. Up to the present no strain of mice has been found which is resistant or susceptible to all types of tumour growth. There is no correlation between resistance to subcutaneous grafts of sarcomas 37 and 180 and susceptibility to induced sarcoma. In an earlier paper (202) the occurrence of hepatoma in C,H mice following subcutaneous injection of 1 :2 :5 :6-dibenzanthracene was recorded; such tumours have now been found to occur spontaneously in a certain number of older mice of this strain. In a subsequent paper (384) Andervont gives a preliminary account of hybridisation experiments with strains C,H, I and Y in relation to the incidence of subcutaneous and pulmonary tumours induced by 1 :2 :5 :6-dibenzanthracene. Some of these results support the extrachromosomal theory of the origin of spontaneous mammary cancer in mice. Shear (793) records, among other results in mice, (1) the production of tumours (apparently sarcomas) with 10-methoxy-l : 2-benzanthracene; (2) the occurrence of liver-cell tumours after treatment with 2-amino-anthracene with the object of producing bladder tumours; (3) tumours of the brain and of the spleen following implantation of pellets of methylcholanthrene “ with the pellets intact in the interiors of the tumours induced ”; (4) failure to obtain tumours with triphenylbenzene even after twenty months. Craciun, Zugravesco, and Stefu (480) report the occurrence of two tumours in mice receiving subcutaneous injections of gamma-methyl-cyclo- 526 J. W. COOK AND E. L. KENNAWAY

TABLEXIV: Dibcnsonthraune Tumors in Micc of Non-cancer Strains (Dobrovolskaia-Zauadskda and Adamm)

Mice yielding tumours at site of injection Number of Strain mice Number Percentage XVI I nc. 5 8.8 21 23.9 XXXIX 6 25.0 15 31.9 XLI 13 31.0 16 30.2 XLII 3 17.6 5 33.3 XXX 9 18.9 10 18.2 Totals 445 103 23.1 penteno-phenanthrene (“ Diels’ hydrocarbon ”) . One appeared a fortnight after the injection and occupied, in large part, the right hemithorax. The sex. of these mice and the total number injected are not stated, and the description given does not exclude the possibility that these were spontaneous mammary tumours. DobrovolskaYa-Zavadskai’a and Adamova (502) examined the action of 1 :2 :5 :6-dibenzanthracene injected subcutaneously in olive oil or lard upon 5 strains of mice in which spontaneous mammary cancer does not occur. The percentage of mice yielding sarcomas in the various strains is shown in Table XIV. These same workers (503) give a large quantity of data, which should be consulted in the original paper, upon the occurrence of tumours in 1184 mice of strain R 111, of which some were untreated while others were treated with tar, radon, or 1 :2 :5 :6-dibenzanthracene (the statement that some of the mice received 1: 2 :3 :4-dibenzanthracene appears to be an error), The hydrocarbon was injected subcutaneously in the left axilla, on a single occasion, in amounts of 0.0025 to 0.1 mg. The incidence of mammary tumours in untreated females was 53.5 per cent and in those receiving 1 :2 :5 :6-dibenzanthracene 47.6 per cent. No evidence was obtained that the hydrocarbon had any tendency to produce tumours in sites remote from the injection. No primary cancer of the lung was observed. (The authors do not mention the adenomas of the lungs of mice.) Of 166 males receiving the hydrocarbon, 40 showed malignant tumours, of which 35 were sarcomas at the site of injection. M. R. Lewis (639) obtained 4 lymphosarcomata from among 100 tumours induced in mice during a year’s time by injection of 1 :2 :5 :6-dibenzanthracene (0.8 mg. in olive oil). One of the tumours appeared in a mouse of the CeH strain at the site of an injection made seventy-five days before. It was transplanted into another mouse, and a large tumour developed composed of lymphocytes. This tumour was transplanted through 12 generations of C,H mice in 152 days and retained the character of the primary lymphosarcoma. The tumour failed to grow in 3 other strains. In mice bearing the transplanted tumours the adjacent lymph nodes of CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 527 the axilla and groin enlarged and were invaded by tumour tissue, while the peritoneal and pleural lymph nodes retained their normal structure. The spleen weighed from 0.3 to 1.0 gm. (normal 0.09 to 0.15 gm.) and contained large areas of actively multiplying lymphocytes. “ The blood of the mice bearing the transplanted lymphosarcoma did not exhibit the neutrophilia characteristic of the majority of transplanted dibenzanthracene tumors (Lewis, 294, 295). On the other hand, the blood picture was not that of a lymphatic leukemia. It was difficult to identify the malignant lymphocytes in the stained spreads of peripheral blood, although they must have been present, since normal mice injected subcutaneously with 0.3 to 0.5 c.c of whole blood drawn from the heart of a mouse bearing the lymphosarcoma later developed tumors at the injection site. The tumors that originated from the implanted blood were indistinguishable from those arising from transplanted fragments of the tumor itself.” The malignant lymphocytes were abundant in the spleen and lymph nodes and to a lesser extent in the kidney, lungs, and liver. “ Fragments of spleen and of axillary and inguinal lymph nodes, implanted into normal mice, brought about tumors in about the same length of time as did pieces of tumor.” The tumour was not transmitted by plasma or by centrifugalized extracts of tumour. The primary tumour and grafts of tumour of the various organs were composed of closely packed lymphocytes. In tissue culture these cells grew to a much larger size than did lymphocytes from normal lymph nodes. The mouse bearing the primary tumour showed areas of invading lymphocytes in the kidneys, lungs, liver, and spleen. The spleen weighed only 0.2 gm. A second mouse of strain CnH which “had received a small amount of dibenzanthracene into the pleural cavity 115 days previously ” showed a lymphosarcoma of the thymus which invaded the lungs and heart and almost destroyed the muscular structure of the auricles. About two-thirds of the lung was filled with tumour. The spleen was not enlarged. The tumour was found post mortem and no grafts were made. “A third lymphosarcoma developed from the lymph node at the site of the dibenzanthracene injection in the right axilla of a BA strain mouse and spread through the muscles and subcutaneous tissue of that region. This mouse had received two injections of dibenzanthracene in olive oil 237 days previously. One injection made into the right axilla resulted in the lymphosarcoma, the other made on the back at the base of the tail gave rise to a large spindle-cell sarcoma.” There were areas of lymphoid cells in the lungs, spleen, and kidney. A fourth mouse (C,,H) showed a transplantable tumour apparently composed of lymphoblasts arising from a mediastinal lymph node. The site of injection is not stated. Thus of these 4 tumours 2 arose in the immediate vicinity of the injection. Barnes and Furth (413) injected 3:4-benzpyrene (1 mg. in lard) into the spleen of a mouse aged one month. Seven and a half months later the following differential blood count was obtained : polymorphonuclears 53 per cent, lymphocytes 14 per cent, abnormal cells, 33 per cent. These last cells proved to be the malignant cells of this strain. They were larger than normal leuko- cytes (the diameter being 11 to 17.5 p, average 13.4 p). The nucleus, which 528 J. W. COOX AND E. L. KENNAWAY was of very varied and irregular shape, filled the greater part of the cell. A large part of the spleen was replaced by these cells, which showed numerous mitoses. The pulp also contained megakaryocytes and immature myeloid cells. “ The greater part of the lymph node that was examined was occupied by dense masses of the malignant cells,” and the bone marrow was almost completely replaced by them. The disease was found to be transmissible by intravenous injections of a suspension of cells from “ leukemic tumor tissues,” but the site of this latter tumour is not clear. ‘‘ The disease produced by intravenous inoculation had the features of leukaemia without tumour formation. . . . In some mice the structure of the spleen was entirely obliterated by the invading cells. . . ,” The lymph nodes and bone marrow were largely replaced by malignant cells, Following subcutaneous injection tumours appeared at the site of inoculation after four to seven days, and these were composed of the malignant cells. Transmission by cell-free material was not ac- complished. The disease was readily transmitted to mice of the same stock, while inoculated mice were rendered susceptible by massive repeated doses of x-rays, but not by a single irradiation. J. and 0. B. Furth and C. Breedis (549) injected about 0.5 C.C. of 4 per cent 3:4-benzpyrene in lard into the spleen of mice, an equal number of closely related mice serving as controls (96 injected and 95 controls). SpZenk turnours appeared in 5 of the injected and in none of the control mice. In 2 of the former large tumours appeared at the site of injection four and eighteen and a half months later, destroying the spleen almost completely and invading the surrounding tissue; the tumours consisted of polymorphous cells with very large nuclei and grew in graft for at least one generation. In a third mouse a sarcoma developed, but there was doubt whether it arose in the spleen or in the overlying muscles, In 2 other mice there were infiltration at the site of injection and massive infiltration of the spleen with cells resembling histiocytes. This tumour grew in successive grafts and has been described by Barnes and Furth (413). Lung trrmours occurred in 29 per cent of the inoculated and in 9 per cent of the control mice. These tumours were multiple in 3 of the 9 controls and in 21 of the 28 injected mice. The microscopic structure was similar in both sets of mice. In about 9 per cent of the mice that received intrasplenic injections of benzpyrene at the age of one and a half to five months, there was an overgrowth of mononuclear cells, mainly in the liver and to a lesser extent in the spleen and lymph nodes. Small tumour nodules formed by these cells occurred in the liver and spleen. In these nodules a neoplastic disturbance of histiocytes with some of the characteristics of Hodgkin’s disease was evident; this did not occur in the control mice. Myeloid leukaemia was more frequent among the injected mice than among the controls, but further data are needed upon this point. M. R. Lewis (641) describes a transmissible monocytoma which she obtained by transplanting a white spot on the spleen of a mouse injected intraperitoneally With 0.8 mg. of 1:2 :5 :bdibenzanthracene in olive oil 41 1 days previously. A full account of the cytology of the tumor and of grafts is given. Mice bearing subcutaneous grafts showed enlargement of the spleen. Metas- CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 529 tases occurred first in the liver, later in the spleen, thymus, and bronchial lymph nodes. Tumours resembling the original monocytoma were obtained when fragments of metastatic tissue from any organ were grafted, and monocytoma developed from fragments of liver and of spleen that did not exhibit macroscopic metastases. The tumour was transmitted by subcutaneous injections of whole blood, but not of plasma. The cells of the tumour were phagocytic and ingested red blood cells, cell dCbris, and bilirubin crystals. At- tempts to transmit the tumour by cell-free filtrates gave negative results. Morton and Mider (696) applied methylcholanthrene (0.5 per cent in benzene) to 9 different areas of the skin of 48 dilute brown mice in such a way that the same area was not painted twice during the same month. After the tenth painting (69th day) 5 female and 5 male mice developed a condition characterized by (1) great enlargement of axillary, inguinal, abdominal and tracheo-bronchial lymph nodes, which were filled with atypical large lymphoid cells with large nuclei and numerous mitoses: (2) enlarged spleen in which the pulp was replaced by cells similar to those found in the lymph nodes; (3) ‘‘ leukemic infiltration ” of lung, liver, skin, coagulating gland, bladder, kidney, and skeletal muscle; (4) replacement of the myeloid cells of the bone marrow by lymphoid cells; (5) secondary anemia. In two mice the white cells numbered 90,000 and 139,000, almost all being lymphoid cells. In 5 mice which developed skin tumours there was no lymphadenopathy. No tumours appeared in 50 control mice. In this strain “leukemia has occurred rarely and never before the animals were eighteen months old (C. C. Little, personal communication) ,” Klinke (608) injected 3:4-benzpyrene (1 per cent in a mixture of lard and olive oil) into the thigh muscles of mice and rats and obtained a large number of sarcomata, of which the structure, chiefly polymorphous and spindle-cell, is described. No neoplastic growth of muscular tissue was found. Brunschwig and Bissell (450) injected 3 :4-benzpyrene either as crystals or in liquid paraftin, or in cylinders of cholesterol, into the femur in mice and rats. In one mouse, in which benzpyrene in cholesterol had been inserted into the tibia as well, an osteosarcoma developed after eight and a half months at the site of injection. Fibrosarcoma of the femur was produced in a rat in which a pellet of methylcholanthrene and cholesterol had been inserted into the bone. In a later paper Brunschwig (449) describes experiments in which methylcholanthrene crystals in a paraffm pellet were introduced into the medullary cavity of either the tibia or femur of 33 rats, of which 4 developed fibrosarcoma of the tibia. Thus of 5 induced primary sarcomas of the bone 1, in a mouse, was an osteosarcoma, while 5, all in rats, were fibrosarcomas. Sutro and Pomerantz (813) injected 1:2 :5 :6-dibenzanthracene in benzene or lard in or near bones or joints in 35 rats, and reproduce 27 photographs and roentgenograms of the tumours--chiefly “ cellular fibrosarcomas ”which appeared in 16 animals. No osteogenic sarcomas were obtained. The tumours did not show any strong tendency to invade the bone and there was little periosteal reaction to contiguous tumours. “. . . most of the spleens, especially those of the tumour-bearing rats, showed definite hyperplasia of the reticulum cells. The haemopoietic cells were also hyperplastic.” Sarcomata were induced by Rothbard and Herman (776) by intramus- 530 J. W. COOK AND E. L. KENNAWAY cular injection of 3:4-benzpyrene in lard in 3 fowls, of which 2 showed metastases. Only one of these tumours could be transplanted by intramuscular inoculation and none could be propagated by injection intravenously or into the anterior chamber of the eye, nor by implantation upon the chorio- allantois, though the virus-induced sarcoma 13 of Stubbs and Furth was readily transmitted by these methods. Peers (739) introduced 1:2 :5:6dibenzanthracene (0.072 mg. in cholesterol) within the parietal region of the skull in mice, of which 53 lived for more than six months. There was very little reaction in the skull or meninges. In one mouse a spindle-cell sarcoma was discovered on the 282nd day on the external surface of the skull over the point of inoculation. No other tumours were found which could be attributed to the hydrocarbon. Seligman, Shear and Alexander ( 788) implanted methylcholanthrene intracerebrally in 20 CJH mice, the pellet being pressed through the dura into the cerebral cortex in the parietal region. There were no perceptible ill effects from the operation. Eleven mice developed gliomas, which were of several types, and two fibrosarcomas were produced which probably arose from the meninges. One glioma and one sarcoma were transplanted in the same strain, and these tumours at the time of the report had reached the 5th and 11th grafted generations respectively. Methylcholanthrene pellets implanted subcutaneously in CsH mice produced sarcomas which appeared more rapidly than did the induced brain tumours. Subarachnoid injections gave negative results. This paper is illustrated by 68 photographs. Weil (839) also studied the effects of hydrocarbons injected intracerebrally. He injected into the brains of rats, numbering 20 in all, ( 1I) 1: 2 :5 :6-dibenzanthracene in lard and in cholesterol; (2) methylcholanthrene in lard; (3) lard or cholesterol alone; (4) styryl430 in water or saline. The dibenzanthracene, methylcholanthrene, lard and cholesterol produced either granulomas or nothing, except that one rat receiving 1: 2 :5 :6-dibenzanthracene in lard showed, in addition to granuloma, a malignant tumour within the brain resembling a squamous epithelioma, of which the origin could not be determined exactly. In the last 3 of 5 rats receiving styryl 430 there were found, after about four months, tumours in every tissue with which the compound came in contact, namely subcutaneous sarcoma, glioblastoma, meningioma of the pia arachnoid, and tumours of the ependyma of the ventricles, and " neoplastic transformation took place in the pars nervosa and glandular portion " of the hypophysis. Oberling, SanniC, and GuCrin (72 7), whose earlier experiments (319) suggested that hypophyseal tumours could be produced by 3 :4-benzpyrene, now question this conclusion in view of the frequency of spontaneous tumours of this type in old mice. Athias and Furtado Dias (397) injected rnethylcholanthrene (2 injections of 0.4 C.C. of 0.3 per cent in arachis oil) into the left kidney of 22 rats. Three of these, which died 173, 177 and 211 days after the second injection, showed tumours occupying almost the whole abdominal cavity. The tumours were polyrnorphous sarcomas showing very numerous normal and abnormal mitoses. In one rat there were metastases in the lung. The kidney tissue was almost completely destroyed. One tumour showed rapid growth in successive transplant generations. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 531

Magnus (660) introduced 1: 2 :5 :6-dibenzanthracene in olive oil by a blunt needle into the stomachs of 100 stock female mice twice weekly for six months, giving a total dose of 104 mg. Of 63 mice alive at the end of this period 60 “ died with pulmonary turnours,” of which 75 per cent were considered to be malignant; in 2 animals there was a metastasis (liver, adrenal). The author considers it most probabIe that the hydrocarbon reaches the lung by the trachea. No tumours of the alimentary tract were observed. One of 25 control female mice showed new growth in the lung, namely multiple benign adenomata. Van Prohaska, Brunschwig and Wilson (753) with a glass dropper gave methylcholanthrene (1 per cent in olive oil) by mouth to mice. Of 48 animals observed 186 days, (a) 2 showed squamous papilloma of the fundus of the stomach, and one of these a carcinoma of the outer surface of the cheek also; (b) 4 showed carcinoma, and 2 papilloma, of the hypopharynx, inside of the mouth, or in portions of the skin adjacent to the mouth. Negative results were given by feeding 1 :2 :5 : 6-dibenzanthracene in lard to mice three times weekly for six months. (Cf. Waterman, 359, 360.) Nicod and Regamey (719) have published extensive data upon the action of 3 :4-benzpyrene and methylcholanthrene upon mice, when given by application to the skin or injection subcutaneously, intramuscularly or into the peritoneum. Injection of methylcholanthrene into the scrotum or testes of 30 mice produced one sarcoma only, and administration with the food produced no tumours. The only tumours obtained, therefore, were carcinomas of the epidermis and sarcomas of the connective tissues. The authors figure examples of adenomas of the lung occurring in mice receiving intrathoracic injections of methylcholanthrene and seem to be unaware of the very frequent occurrence of such tumours in these animals. Strong, Smith, and Gardner (808) applied to mice of the CBA and A stocks 3 :4: 5 :6-dibenzcarbazole, which has been shown by Boyland and Brues (219) to produce neoplastic growth in the liver, as well as epithelioma of the skin and sarcoma. The CBA strain is liable to spontaneous carcinoma of the mammary gland and lung, ovary and skin, to melanoma, giant-cell sarcoma, and hepatoma. At the time of this report 42 mice of this strain had been observed to develop spontaneous hepatomas at an average age of 613 days, males and females being about equally affected. A variety of liver-cell types (flattened, polygonal, oval, round, and spindle-shaped) were present in these tumours, but the bile ducts appeared to be absent. The A strain shows carcinoma and adenoma of the mamma and of the lung, embryoma, and leukaemia, but no spontaneous hepatomas have been seen. In the CBA strain the application of dibenzcarbazole to the skin in benzene produced squamous-cell carcinomas of the skin and sarcoma and hyperplasia of small bile capillaries. Subcutaneous injection of the compound (0.2 C.C. of 0.1 per cent in sesame oil until 1.2 mg. had been given) produced spindle- cell sarcomas at the site of injection in 14 out of 18 mice, while 5 mice developed liver-cell tumours similar to those arising spontaneously, but these appeared at dates earlier than do the usual spontaneous hepatomas. At the beginning of the experiment the average age of the 18 mice was 316 days, 532 J. W. COOK AND E. L. HENNAWAY of the 5 mice which developed hepatoma 390 days, and of those mice which developed only sarcoma without liver hyperplasia 288 days. In a third experiment 24 male CBA mice received subcutaneous injections and all developed sarcoma at the site of injection, while 3 showed well formed nodular hepatomas. Eighteen male mice of the A strain received a total of 1.2 mg. of dibenzcarbazole in sesame oil in 6 injections at seven-day intervals. All but one mouse, which was killed at an early stage, showed subcutaneous sarcoma. Shrinkage and thickening of the lobes of the liver occurred, but no hepatomas. Andervont (390) tested the action of 4’-amino-2 :3’-azotoluene (10 mg. injected subcutaneously monthly for eleven months) upon the livers of mice of three strains (A, C, C,H, Y, I). Strain C appeared to be most, and strain Y least, susceptible to the action of the compound. Strain CsHshowed a low susceptibility, although mice of this strain have a high incidence of spontaneous hepatoma; hence in this instance the carcinogenic agent introduced from without does not act with the natural factors to produce a cumulative effect (cf. the lung). Waters (837), in an experiment begun in 1935, confirmed Yoshida’s results by obtaining liver tumours in 15 out of 22 rats living for more than 200 days on a diet containing 4’-amino-2:3’-azotoluene. The least amount of the compound eaten by a rat showing a macroscopic tumour was 3.5 grams. Nakamura et (21. (709) carried out repeated intravenous injections of butter yellow and 4’-amino-2:3’-azotoluene in rats and rabbits over periods up to 260 days, when as many as 33 injections had been given. No neoplasms of the liver were produced. Data are given also on the rate of excretion of these compounds in the urine after the. injections; this rate is more rapid in the rabbit than in the rat. Nakahara and Fujiwara (704) continued their earlier work (313, 314) by injecting p-dimethylaminoazobenzene (butter yellow) in olive oil into the peritoneum of 57 rats once weekly. Six rats lived more than 250 days and had received from 749 to 860 mg. of the compound; typical liver cancers, of which one invaded the diaphragm and another caused metastasis in the lung, were found in three of these. The malignant change in the liver was preceded by cirrhosis; no cholangiomatous growth occurred. No malignant change was observed in the peritoneal cavity nor in organs other than the liver. Nu- merous figures of the tumours are given. Oberling, Gukrin and Gudrin (724) introduced a small fragment of 3:4- benzpyrene into the liver in each of 10 rats. Eight of these lived for more than fourteen months and in one which died at the sixteenth month a histiocytic sarcoma of rare polymorphous character was found in the liver in the region where the inoculation was made. This tumour had been carried on by graft to the fifth generatiodad metastasis occurred in several of the grafted animals. A second rat died in the twenty-fifth month with a histiocytic poly- nodylar sarcoma of the liver 2 cm. in diameter and a monocytic sarcoma re- placing almost the whole of the right lung. Insertion of a crystal of 3:4- benzpyrene into the kidney in 5 rats gave in one at the tenth month an “ kpi- thkliome malpighien kbratinisant ’’ which wetransplanted for two generations. Askanazy (396) describes the following turnours: in rats (1) various sar- CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 533 comata produced by intraperitoneal or subcutaneous injection of 1: 2 :5 :6- dibenzanthracene, 3 :4-benzpyrene, or methylcholanthrene, and (2 ) intracranial grafts of a subcutaneous sarcoma produced by 3 :4-benzpyrene; in rabbits (3) a chondroma developing in foetal tissue, introduced within the cranium, into which 3:4-benzpyrene in olive oil had been injected, and (4) a chondro- sarcoma arising from the dura mater after, intracranial ‘injection of 3 :4- benzpyrene in beef fat. Gye and Foulds (566) found that the golden hamster (Cricetus auratus) is highly susceptible to the production of sarcoma by subcutaneous injection of 3:4-benzpyrene (0.4 per cent in lard). Tumours showed cells of various types, spindle, rounded, multinucleated, and giant cells with a single nucleus. The tumours were transplantable and metastases in the lung occurred in animals bearing both primary and grafted tumours. (Cf. Halberstaedter, Am. J. Cancer 38: 351, 1940.) Rlinke (606) applied 3:4-benzpyrene (1.0, and later 0.5 per cent, in chloroform) twice weekly to the pinna of 50 rabbits, of which 37 died within two months showing in many cases changes in the liver (necrosis, fibrosis, bile- duct proliferation) which were less distinct or absent in those living for longer periods (cf. Claude, 227; Polson, 183). One rabbit which died on the 587th day bore an infiltrating epithelioma. In this report and a later one (607) Klinke describes an experiment in which 3 :4-benzpyrene (as (‘Salbe,” medium not stated) was injected into the thigh muscles of 42 rabbits, of which 15 lived more than 200 days. Between the 224th and S5lst day, sarcomas were obtained at the site of injection in 7 rabbits; one of these had received 460 mg. of the hydrocarbon. The polymorphous cellular structure of the tumours is described; two had formed metastases: in the lung and spleen in one instance and in lung, spleen, kidney and adrenal in the other. Schurch (786) applied 3:4-benzpyrene (0.5 per cent solution; solvent not stated) three times weekly to the ear of a rabbit. Warts appeared on the painted area after eleven months and a squamous epithelioma developed after three years. During the first half of the fourth year “oestroglandol” was injected twice weekly. A large fibrous tumour developed on the ear after three years but retrogressed completely following injections of oestroglandol. At the end of three and a half years’ painting, and after six months’ injection of oestroglandol, a melanoma developed upon the ear, with extensive metastases in the lymph nodes, lung, liver, spleen, and kidneys. Thus a large fibroma, a squamous epithelioma, a malignant melanoma, and numerous papillomas developed upon the same ear. Continuing earlier work with Winterstein ( 120) Schurch (787) applied 3:4-benzpyrene (0.5 per cent, ?‘sqlvent) three times weekly to the ears of 20 rabbits, and obtained precancerous warts in an average of eleven months, and carcinomas in 11 animals after periods varying from thirteen months to three and a half years (average two years and three months). Carcinoma occurred in 80 per cent of those reaching the latter average age. Two large fibrous tumours of the pinna were also obtained. Burrows and Boyland (453) applied 1: 2 :5 :6-dibenzanthracene to the vaginal vault, mammary ducts, and subcutaneous tissues of 36 rabbits over periods up to 959 days. They obtained no tumours at the site of application, 534 J. W. COOK AND E. L. KENNAWAY but uterine tumours appeared in 10 of 16 females which survived for 900 days or more. The question whether these tumours are to be attributed to the action of the hydrocarbon is to be investigated in further experiments which will include sufticient controls. (Cf. Pourbaix and Denisoff, 184; Lambret et d., 288; Leroy et at., 637; Haagensen and Krehbiel, 60.) The literature relating to uterine cancer in the rabbit has been reviewed by Orr and Polson (730), who have added 5 new cases of their own; 2 of the 5 animals had received a very small dose of 1:2:5:h-dibenzanthraceneJ to which the authors do not attribute any significance. Leroy, Randel and Brunschwig (637) injected 1:2 :5 :6-dibenzanthraceneJ 3 :4-benzpyrene, or methylcholanthrene, in suspension (0.3 to 0.5 mg. per c.c.; Boyland’s method, 16) intravenously into 53 rabbits, 5 C.C. being given at intervals of one to three weeks until 20 to 30 mg. had been given. Thirty-one rabbits lived for periods from twenty-six to eighty-one weeks, when the experiment was terminated. No tumours were found in any of these animals. Sabrazb, Bideau and Geyer (779) describe the formation of arthrophytes in the knee-joints of rabbits after injection into the joint cavity of 3:4- benzpyrene in benzene. Control injections of benzene are not mentioned. No neoplastic changes were observed. Valade (825) compared the action of methylcholanthrene and 3 :4-benzpyrene in the production of subcutaneous sarcoma in the rat. They were found to be about equally active and 3 mg. is considered to be the optimum dose. The tumours appeared between forty days and two months. Metastases in the lung occurred frequently; in some areas of these there were dnormes pZus- modes multinucldes.” Subcutaneous injection of these hydrocarbons in 6 dogs produced no tumours in six months, and from this the author concludes that the subcutaneous tissue of the dog is refractory.’ Injections into the mammary gland in 2 female rabbits gave negative results. An injection into the liver of 5 rabbits of an oily solution of 3 :4-benzpyrene produced no symptoms in these animals during four months. Administration of methylcholanthrene in lard in the food to 20 rats twice weekly for four months produced no tumours. Hueper, Wiley and Wolfe (587) injected beta-naphthylamine in water subcutaneously daily into 16 female dogs in doses of 4 to 5 mg. per kilo for about four months; the dose was then doubled for three months, and thereafter tripled for fifteen months. Parenteral administration was supplemented by feeding (0.1 to 0.45 gm. daily) for various periods. Four similar dogs served as controls. In 13 out of the 16 dogs treated for twenty to twenty-six months various degrees of neoplastic change were found in the bladder, namely (a) benign papillomata; (b) “ papillomata with histological benign and malignant component parts ”; (c) papillary and infiltrative carcinomas. No metastases were found. In some of the dogs the liver showed “ adenomatoid proliferations of liver cells.” Swollen and lactating mammary glands, sometimes with abundant secretion, were observed in many of the dogs, especially in the later period of the experiment. The subject of occupational and experimental tumours of the bladder has 1Passey (J. Path. and Bact. 47: 349, 1938), however, obtained turnours of the skin in dogs after application of tar weekly for six years. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 535 been reviewed by Hueper (Arch. Path. 25: 856, 1938. For recent literature upon occupational cancers of the bladder, see a series of papers in J. Urol. 38: 212-250, 1937). Berenblum and Bonser (422) obtained negative results in an attempt to produce tumours of the bladder in rats and rabbits by intraperitoneal injection, feeding, or inhalation of aniline, alpha or beta naphthylamine, benzidine, or 5-chloro-o-toluidine hydrochloride. Hueper, Briggs, and Wolfe (586) also failed to produce neoplastic changes in the bladder by daily intraperitoneal or intravenous injections of 3 mg. beta- naphthylamine hydrochloride to 15 rabbits for nine months. Five of the 10 females showed signs of pseudo-pregnancy, which the authors ascribe to stimulation accompanying cystoscopy, and considerable development of the mammary glands (cf. Hueper, Wiley, and Wolfe, 587). Duran-Reynals (511) injected into the thigh muscles of 261 frogs of 3 species and 18 newts of 2 species, 1 :2 :5 :6-dibenzanthracene, 3 :4-benzpyrene, or methylcholanthrene in various media; no tumours were obtained and the incidence of LuckC's adenocarcinoma of the kidney was not increased. Koch and G. and B. Schreiber (609) report the results of subcutaneous injection of 3:4-benzpyrene in olive oil in amphibia, namely two species of Triton. The authors consider that malignant changes occurred and describe what they regard as metastases in the lungs. The paper is illustrated by some unsatisfactory photomicrographs. Levine (638) applied Scharlach Red in ether to decapitated shoots of the plant Kalanchoe and obtained overgrowth with leafy shoots and roots resembling typical crown galls induced by P. tumefaciens. No such tumours were produced by 1:2 :5 :6-dibenzanthracene, 3 :4-benzpyrene, methylcholanthrene, or heteroauxin. Abercrombie (375) describes and figures the evocation of a neural plate in the chick embryo by sodium 1 :2 :5 :6-dibenzanthracene-9 : lo-endo-ap- succinate; previously this compound, and 1: 2 :5 :6-dibenzanthracene, which is also active in the chick, had been tested only on Amphibia. " Styryl430, very active in Amphibia, was only slightly more active than plain albumen." No specificity is claimed for these reactions, since " a probably impure specimen of naphthalene, of moderate activity in Amphibia, was the most active substance tried. . . .,, Waddington (830) examined the action of several carcinogenic and oestrogenic substances, and other related compounds, for evocator capacity by implantation in the blastocoele 'of newt gastrulae. " Inductions were obtained with a considerable number of substances, and no hypothesis can yet be made as to the relation between chemical structure and evocator power." Shen (797) found that 1:2 :5 :6-dibenzanthracene-9 : lO-endo-n/Asuccinic acid induces neural tube formation in gastrulae of Triton alpestris, and suggests that the fact that there is an optimal dose (0.0125 y per embryo under stated conditions) indicates that the compound acts directly upon the ecto- dermal cells, as would the natural evocator, and does not merely liberate a masked evocator by a non-specific toxic action. Hall and Franks (574) give a preliminary account of the effect of prolonged administration of acetylcholine upon dogs, guinea-pigs, rats, mice, and 536 J. W. COOK AND E. L. KENNAWAY

TABLEXV: Results of Oestrogen Injedions in Mue (Lacussap)

Mice receiving injections of oestrogens Dead without Dead with cancer cancer 80 dQ Strain R3...... 6 9 32 21 Mammary cancer...... 51 Mammary cancer with lymphosarcoma of thymus...... 1 Spindle-celled subcutaneous sarcoma and glandular lymphosarcoma...... 1 Strain R4and 17...... 3 2 63 Mammary cancer...... 63 Strain 17 nc...... 23 12 31 Lymphoearcoma of thymus...... 1 Spindle-cell subcutaneous sarcoma...... 11 Adenocarcinoma of the pituitary...... 1 Strain 30 ...... 14 14 47 Lymphosarcoma of thymus...... 31 Glandular lymphomcoma...... 13 Spindlecell subcutaneous aarcoma...... 1 Adenocarcinoma of the thyroid...... 1 Sarcoma of bladder...... 1 Strain 39 ...... IS 12 33 Lymphosarcoma of thymus...... 31 Epithelioma of uterus...... 1 Epithelioma of lung...... 1 TOTALS...... 61 + 49 - 110 66 + 49 - 115 fowls. “ An inordinate number of tumors have developed in various animals. Most are definitely malignant, some. carcinomatous. None occurred at the site of the prolonged daily acetylcholine injections.” No details of the tumours are given.

IIZ. Oestrogens in Relation to Tumours Lacassagne (621, 621A, 622) has published valuable reviews upon the relationship of hormones and mammary adenocarcinoma in the mouse, in which data are brought together upon the action of the pituitary and of testosterone, together with other matters. He has reported, also (619), a series of experiments in which he injected various oestrogens (oestrone, oestradiol, equilin, equilenin as benzoates) and mixtures of these, subcutaneously, in oily solution, into mice, beginning a few days after birth. Mammary adeno-’ carcinoma developed only in strains in which these tumours occur spontaneously, but in these strains mammary tumours appeared in the injected mice more often and earlier than in controls and as frequently in males as in females. Sarcomas and epitheliomas were observed in strains in which they were known not to have occurred spontaneously for several years, from which the author concludes that the oestrogenic hormones played a part in their production. The results of his experiments are summarised in Table XV. Lacassagne and Raynaud (629) enumerate the changes in various organs CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 537 produced in mice of the RIII strain by oestrone benzoate, by testosterone propionate, or by both together, and give details of experiments in which mice of this strain receiving various amounts of these two compounds, either simul- taneously or successively, developed mammary cancer. Amounts of testosterone which would be sufficient to lessen considerably the development of the mamma in male mice receiving oestrone alone did not prevent the development of mammary cancer in mice receiving both compounds. For details of the amounts given, the original paper must be consulted. The same authors (628, 629) discuss the mode of action of testosterone upon the development of mammary cancer, and give further data on the antagonism of oestrone and testosterone and on their comparative action upon different organs. Tomita (818) examined the effects of a male hormone preparation upon the development of liver tumours in rats receiving 4’-amino-2 :3’-azotoluene by the mouth. The hormone had a considerable accelerating action upon the development of tumours in male but not in female rats. “ Cholestedn- InjeRtion” had a slight effect of the same kind. Extensive data are given upon the weights of the body and of various organs of these animals, for which the original paper must be consulted. Maisin, Pourbaix and Rijckaert (668) injected testosterone propionate in oil (? nature) every fifteen days for 134 days into two series of mice receiving applications of 3:4-benzpyrene to the skin three times weekly. One series received in all 0.9 mg. and the other 0.09 mg. of the propionate. The former showed a possible slight inhibition of carcinogenesis; otherwise no effect was noted except greater length of life in comparison with controls. No tumours appeared at the site of injection during 360 days of observation. Nathanson and Andervont (7 11) injected testosterone propionate (0.5 mg.) in sesame oil three times weekly into 20 (A) out of a batch of 40 female C,H mice which previously had had, at maturity, one litter which was killed within twenty-four hours. The remaining 20 (B) received control injections of sesame oil only. Only 6 of the A mice developed mammary carcinoma; the tumours were single and grew at the normal rate. Four animals died in the twelfth month without tumours and 10 were alive at the sixteenth month, all without tumours. In group B every mouse had developed from one to five mammary tumours by the eleventh month, and these tumours caused death by the fourteenth month. Further experiments upon 40 female C,H mice already bearing mammary tumours showed no difference between those treated with testosterone propionate in sesame oil and those injected with the oil alone. Stewart (805) has reviewed the contradictory literature upon the effect of the gonads and sex hormones on carcinogenesis. In his own experiments he found that ‘‘ castration decreased slightly, but not apparently to a significant extent, the average time of development of tumours in male and female mice of the C,H strain injected subcutaneously into the right axillary region with 0.8 mg. of 1:2:5:6-dibenzanthracene in 0.2 C.C. of lard. However, up to the end of the sixteenth week a higher percentage of tumours appeared in the castrates of each sex.” Lacassagne (620) gives data on the amount of testosterone required to prevent the development of mammary cancer in mice of the RIII strain (normal incidence 60 to 70 per cent in females). In two male mice receiving in 538 J. W. COOK AND E. L. KENNAWAY alternate weeks 50 y of oestrone and 0.5 to 1 mg. testosterone, the latter did not seem to exert any antagonistic effect on the breast. Mammary tumours appeared after the usual interval and developed normally. The usual changes induced by oestrone in the coagulating gland, however, were absent in one and slight in the other mouse. In RIII females mammary cancer developed normally in those receiving 0.5 to 1 mg. testosterone acetate weekly, but was absent in those receiving 1 mg. of the propionate twice weekly. Lacassagne considers that testosterone in the female mouse acts not directly on the mammary gland, but on the ovary through the pituitary. He describes the structure of the ovary in mice receiving testosterone. The anterior pituitary under the action of oestrone shows hypertrophy, sometimes adenomata, congestion, much colloid, and scarcity of acidophil cells; under the action of testosterone the pituitary is small and contains many acidophil cells. Flaks and Ber (542) injected testosterone propionate (" sterandryl ") into mice during the application three times weekly of methylcholanthrene to the skin, the sterandryl being administered over a period of 138 days and the methylcholanthrene for 154 days. The results are shown in Table XVI.

TABLE XVI: Effect of Testosterone Propionate on Development of Tumours Foll~whgAdminisIra- Lion of Methylcholanthrene: Exfierimnt I (Fluks and Ber)

Number of days Mice bearing tumours of experiment Controls Receiving testosterone 0 0 0 90 16.1% 7.9% 120 41.1% 29.4% 150 69.6% 54.9% 190 76.8% 70.6%

In a second experiment, in which 51 mg. of testosterone propionate was given to each mouse, the result shown in Table XVII was obtained. Ster- andryl was given for 144 days and methylcholanthrene for 142 days. The number of mice was limited by the supply of testosterone.

TABLEXVII: Efed of Testaskrone ProQiorurk on Development of Tumours Following Adminis- tration of Methylcholanthrene: Experiment 11 (Fluks and Bcr) -~ Controls Experimental Group

Number Total Number of Number Total Number of Number of of number sarcomas of number sarcomas days of mice of among mice of among experiment living tumours tumours living tumours tumoura 0 10 0 0 10 0 0 130 10 7 0 0 t 0 150 10 9 4 9 3 0

In a continuation of these experiments Flaks and Ber (543) found that testosterone injected into mice during the application of methylcholanthrene or 3:4-benzpyrene to the skin did not affect the incidence or time of appearance of papillomas, but delayed the development of cancers by as much as two months. About 100 mg. of testosterone per mouse was required to produce this effect. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 539

Amano and Tomita (378), continuing the work of Tomita (350), gave weekly injections of ‘‘ male hormone ’’ (“ androstin ”; the compound contained in this preparation is not stated) to a group of rats receiving 4’-amino-2:3’- azotoluene in the food: another group received cholesterol in the food in addition to 4’-amino-2 :3’-azotoluene, and a third group was untreated except for the amino-azotoluene. The rats were killed on the 270th day. The results are shown in Table XVIII. These are somewhat confusing as the proportions of the sexes in the 150 rats are not stated. The liver of one of the male rats weighed 60 gm. None of the female rats in any of the 3 groups showed hepatoma. The body growth was checked, especially in the group treated with male hormone. The authors are inclined to correlate their results with the greater prevalence of cancer of the liver in men than in women in Japan.

TABLEXVIII: Male Hormone and Hepatoma Formation (Amano and Tomila) ___ Number of rats after Nodular Initial 270 days hyperplasia Adcnoma Hepatom:i -~ -~ Androstin group SO 10 2 1 7 Cholesterol group SO 6 2 2 2 Controls 50 3 3 0 0

Using a small number of rats, Ito (593A) compared the incidence of liver tumours after feeding with 4’-amino-2 :3’-azotoluene (310 days feeding followed by 56 days normal diet) in normal and castrated females. Castration appeared to hasten the development of these tumours (Table XIX).

Initial Alive Livern hyperplasiaNodular Adenoma Hepatollla numberrats of 36;fteeys -__ --__ -___ ~ _- Controls 27 15 7 7 3 2 Ovarian castration 21 12 7 7 7 6

Lacassagne (625) found that thyrotropic pituitary hormone did not prevent the development of mammary cancer in RIII mice of either sex which had received the usual dose of oestrone benzoate. Cramer and Horning (482,483) studied the adrenal glands of 95 mice be- longing to six inbred strains, of which three showed no mammary cancer during two or three years’ observation. The results are summarised in Table XX. The authors consider that in the two high-cancer strains the inherited susceptibility to mammary cancer is associated with an impairment of the func- tional activity of the adrenal medulla, which favours the action of the ovarian oestrogens on the mamma. In these mice, the pituitary remains normal. Adrenalectomised mice are resistant to the production of mammary cancer by oestrogens and hence there appears to be a synergism between the adrenal cortex and the ovary, for if even a small amount of cortical tissue were present, in the form of an adenoma, “ the aninlal remained as sensitive tQoestrinisa- tion as an intact animal, if not more so.” In contrasting the medullary with 540 J. W. COOK AND E. L. KENNAWAY

TABLEw(: Brown Degenerasion in Stock Mice and Mia of Inbred Strains (Cram and Horn- ing; Dobrowlskala-Zadskaia and Z$phirob)

Strain Occurrence of Brown degeneration Site of Brown degeneration Cramcr and Horning Stock mice Never observed in males or females up to twelve months: may be prm- ent to slight degree in older mice Stock mice with sponta- In 4 out of 7 females neous mammary cancer Stock mice treated with Present in both males and females In zona reticularis; may ex- estrogens tend to adjacent medulla - R 111 and Dilute Always in R 111 females with mam- Chiefly, but not exclusively, Brown (highcancer) mary cancer and fairly regularly in in medulla; later extending to both males and females of this cortex, but not confined to strain at four to six months of age. zona reticularis In both strains occurs at early age (1 at sex maturity) and at six months was present in all but one mouse of each strain. Males and females of equal ages about eauallv affected. Bagg albino (lowcancer) Did not appear till 14th month, I Simpson (cancer-free) and then not progressive. Absent Black (cancer-free) altogether in Bagg and Agouti Agouti (cancer-free) males Dobrbwlskaib-Zawdskaio and Z6phirof * R I11 (high-cancer) In 5 out of 8 males and in all 48 females examined 5 cancer-free strains Not at all, or only in early stages. Does not accompany Ra or dibenr- ? anthracene tumours

* Compt. rend. Soc. de biol. 128: 971, 1938. the cortical type of brown degeneration, the authors say: ‘‘ The opposite significance attaches to the cortical type of brown degeneration, which is induced in mice of mixed strains by oestrinisation. Under these conditions the hormonal balance is disturbed primarily by an excess of oestrogenic hormone with its carcinogenic effect on the mamma. This induces the cortical type of brown degeneration, which leads to a diminution of the synergic factor and thus repre- sents, together with the change in the pituitary, described previously, an attempt to restore the hormonal balance. These two conditions represent, therefore, two different aetiological types of cancer of the mamma.” The exact mechanism of the ‘‘ attempt ” described above is not made very clear. [On the subject of brown degeneration in the adrenal, see also Dobrovol- skiiia-Zavadskda: Compt. rend. SOC.de biol. 125: 877, 1937; Dobrovolskaia- Zavadskai’a and Zbphiroff: ibid. 128: 971, 1938; Lacassagne and Raynaud: ibid. 124: 1183, 1186, 1937; Twombly, 822.1 Dobrovolskaia-Zavadskalaand Pezzini ( 505) distinguish between degeneration of the adrenals and vacuolisation and pigmentation. The figures for brown degeneration only are quoted here. The ages of the mice are not stated. In 17 males and 18 females of 5 strains exempt from mammary cancer, there CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 541 was brown degeneration in 3 only (sex not stated). In strain RIII brown degeneration was found in 5 of 8 males and in 56 of 60 females. In 13 other high-cancer strains it was present in 6 of 20 males and 45 of 62 females. In the females the incidence of brown degeneration was the same (73 per cent) in 31 having mammary cancers and in 31 having other tumours or free from neoplastic growth. Observations on 10 strains are given in Table XXI.

TABLEXXT: Brown Degeneration of the Adrenals in Mice of Vurious Strains (Dohrovolskala- Zavadskalia and Pezsini) - .- Incidence of mammary Brown degeneration cancer in females Males Females 8 lines: FI, IV, XXIX, 3 per cent to 40 In 9 of 32 In 37 (Total examined 111, with XXXIII, XI., XLIII, per cent 72 mammary cancers, 22 sar- XLIV, XLV comas, 2 epitheliomas, 1 lung cancer) 2 lines: XIX arid 27 per cent and In 1 of 6 In 4: 1 with mammary cancer, 1 XXXVl I I 11.7 per cent with sarcoma, 1 with epithelioma. (Total examined 37, with 24 mammary cancers)

Lacassagne (627) records a number of observations summarised in Tables XXII and XI11 on the incidence of sarcoma in mice receiving injections of oestrogens, allied compounds, and some others. Only animals which had been subjected to treatment for more than 300 days are included, as tumours of the type in question do not appear until after about a year of treatment.

TABLEXXII: Incidence of S~YU)Min Treated Mice of Struin R III (Lacassape) . - Developing sarcoma I Not developing sarcoma Substance Injected Males Females Males Females Testosterone propionate Testosterone acetate 2 Testosterone propionate and 2 oestrone Androstenediol dipropionate 2 Olive oil + 5 per cent ethylene 1 glycol Oestrogens (oestrone, equiiin, equilenin) Progesterone

Table XXII shows that in the high-cancer strain RIII, 13 subcutaneous spindle-cell sarcomas occurred in 45 mice, while 11 of these tumours arose among the 27 mice receiving male hormone. The small number of female mice is due to the removal from the experiment of those dying from mammary cancer before the 300th day. The low-cancer strains llnc, 30, and 39 are less susceptible to the production of sarcomas by the action of such agents. Burns, Suntzeff and Loeb (451) obtained a sarcoma in each of 7 mice injected with " Progynon B " (oestradiol benzoate). The total number of mice injected with this material is not stated. Another sarcoma developed in a 542 J. W. COOK AND E. L. KENNAWAY

'rABLE XXIII: Inc~dmceof Sarcoma in Treakd MU8 of Strains 17nc, 30, 39 (kssagnc) - Developing mrmma I Not developing mrcoma Subtance injected

Testosterone acetate 1 (uterus) 6 2 Testosterone (acetate or propionatr) 7 4 and wstrone Dipropionate of androstenediol 2 Olive oil 4 2 Oestrogena alone or with proges- 1 1 3 (1 bladder) 1 3: I 27 terone or pituitary extract Progesterone 3

TOTAL- 11 141.56 I4O mouse receiving injections of " estrogen " and lutein. No sarcomas appeared in 128 non-injected control mice observed over a similar period. All the tumours except one developed at or near the site of injection and appeared after a long period of treatment. Turner (820) injected (a) ox bile ( 1 gm. dry ox bile in 10 C.C. water), (6) oestrone (referred to as estrin), or (c) both these together into mice of two strains, namely CsH (95-100 per cent incidence of cancers of breast in females and about 22 per cent hepatomas in males) and I (5 per cent incidence of

TABLEXXIV: Hepatomas in CIH Male MicG Treakd with Ox Bile and Oestrone (Turner)

I Mice Uvlng to 16 month6 of age Material injected rml- weekly for 34 weka Number of Number of mice Per Cent of mtce mice I withhepatomaa 1 havlnghepPtoma8 Ox bile 9 77 Ox bile and estrin 10 70 Estrin 10 50 Untreated controls 19 26 cancers of breast in females and hyperplasia of gastric mucosa in both sexes). The results are shown in Table XXIV. Injection of bile in C,H mice appeared to increase the incidence of hepatoma. Cf. the diminished incidence of hepatoma in mice receiving liver in the food with butter yellow (Nakahara, Mori and Fujiwara, 708). " It is of interest to note " Turner says, " that, while untreated virgin C,H female mice develop spontaneous breast tumors at an average age of 11.5 months, the ' bile and estrin ' CsH female mice in this experiment developed tumors at an average age of 8.2 months. Also, in the thirty-fourth week of the test, when the treatments were stopped, the mice which had received bile and estrin ' had four times as many mammary tumors as the ' bile only ' group. This suggests that 340 international units of estrin, alone or when combined with 0.136 gm. of bile, lower the average age at which breast tumors occur in virgin CsH females. However, the number of mice is too small to permit definite conclusions in this regard. A male CsH mouse in the ' estrin only ' CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 543 group developed a mammary tumor in the fortieth week, after having received only 340 international units of estrin.” Six types of malignant tumour, namely 2 lymphomas, a carcinoma of the skin and of the breast, a subcutaneous sarcoma, a pulmonary adenoma, and hepatomas, occurred in 33 injected C,H male mice (for details see original paper), whereas in the control mice only hepatomas occurred (Cf. Perry and Ginzton, 323). Lacassagne (624) injected subcutaneously 25 y of diethylstilboestrol in sesame oil twice weekly in a litter of mice (2 males and 3 females) of strain RIII, in which 70 per cent of the females but none of the males have spontaneous mammary adenoma. The 3 females died or were killed in extremis on the 95th, 131st and 139th days. All three showed jaundice, but no macroscopic mammary tumours. The males developed mammary cancers on the 84th and 112th days; these were adenocystic and showed the structure seen in spontaneous cancers or those produced by oestrone. In the male organs there occurred the changes seen after prolonged treatment with oestrone: at- rophy of the testicles, absence of spermatozoa, fibrosis of the vesiculae semi- nales, squamous metaplasia of the coagulating glands. In the females there were leucocytic infiltration of the uterus or pyometra, and keratosis of the vagina. The very early development of cancer in the males is noteworthy. Geschickter (554) produced mammary cancer in 26 out of 86 male and female castrate and non-castrate rats (the table shows 19 females and 6 males = 25) by injections of oestrone. He found a daily dose of 200 y injected intramuscularly suitable for the study of the production of mammary cancer. With this dosage microscopic tumours appeared within 150 to 200 days. When pellets of the crystalline compound were implanted, cancer appeared in twenty-three to fifty days. “ With pellets a total of 5 to 10 milligrams is sufficient, whereas with injections in oil a total of 30 to 40 milligrams is used.” In an experiment still in progress at the time of the report cancer was said to have been ‘‘ obtained in one rat injected with 200 gamma of diethylstilboestrol daily for 100 days.” Robson and Bonser (761) obtained between the eighteenth and twenty- ninth weeks 10 mammary adenocarcinomas in 9 out of 53 male RIII mice receiving a weekly subcutaneous injection of at first 5 mg., then 3 mg., of triphenylethylene (a synthetic oestrogen) . No tumours were obtained in a similar experiment upon mice of both sexes of a strain (black agouti) not normally liable to mammary cancer. Van Heuverswyn, Folley, and Gardner (580) observed mammary growth in male mice receiving various androgens, oestrogens (for details see original paper) and deoxycorticosterone, and found the greatest amount of growth in mice receiving androstenedione, deoxycorticosterone acetate, 9 : 10-dihydroxy- 9 :10-di-n-propyl-9 : 10-dihydro-l :2 :5 :6-dibenzanthracene, or triphenylethylene. ‘‘ It is particularly interesting that the injection of 3 synthetic estrogenic chemicals differing rather strikingly from the naturally occurring estrogens; namely, triphenylethylene, stilbestrol and the dibenzanthracene compound are followed by mammary growth. The mammary growth in mice receiving deoxycorticosterone acetate indicates again an overlap in physiological activity. . . . The slighter mammary response in animals receiving the larger amounts 544 J. W. COOK AND E. L. KENNAWAY of estradiol benzoate and also triphenylethylene and stilbestrol rearms the l stunting effect ’ on mammary tissues of large amounts of estrogens.” Lacassagne and Danysz (623) carried out weekly injections of 50 y of oestrone benzoate fyom the time of birth in mice which were suckled by foster mothers, as in Bittner’s experiments. A male mope of line 39, a line which in seven years has never been observed to produce a mammary carcinoma even under treatment with oestrogens, was suckled by a female of strain RIII and received the injections from the 13th day; it developed a mammary cancer after 182 days of treatment, which is the average period necessary to produce such a tumour by injection of oestrone in males of strain RIII. Twombly (822) placed a single crystal of oestrone, weighing from 0.06 to 0.2 mg. under the skin of each of 21 male mice (RIII) about ten days old. Sixteen of these mice died within 100 days from the results of prostatic enlargement. Of the 5 remaining, 2 developed transplantable mammary carcinomas after about five months. One showed l1tremendous development (both duct growth and acinar proliferation) of the non-cancerous portions of the breast.” Both mice showed brown degeneration of the adrenals and enlargement of the pituitary, in one to twice, in the other to five times the normal size. The author adds a note to the effect that l1since this article was submitted for publication, 13 additional male mice have developed mammary turnours.” Gardner, Allen, Smith, and Strong (552) observed 18 tumours in or near the cervices of mice receiving large amounts of oestrogens (500 international units of benzoate of oestrone or oestradiol every one, two or three weeks over long periods). Most of these growths were of small size and, although they invaded the adjacent mucosa or muscularis, their malignancy could not be ascertained. A malignant cervical tumour was obtained, however, in a mouse (C,H) receiving 500 i. u. oestradiol benzoate every three weeks from the 45th to the 266th day of life (total 5500 i. u.). A mammary tumour appeared at the end of this time and was removed; twenty-four days later a second mammary tumour developed and was also removed. Weekly injections (500 i. u.) were then carried out for seventy-five days (5000 i. u.). The mouse was killed on the 364th day of life on account of a large tumour projecting from the vagina. Pieces of the growth, which arose from the uterus and almost filled the pelvis, were grafted in one female and in 4 male CsH mice and grew rapidly in all, and the tumour at the time of writing was growing in the second generation. The exact origin of the tumour could not be determined on account of its advanced growth, but it consisted of squamous cells similar to those of the smaller tumours mentioned above and invaded the coats of the rectum and bladder. There were metastases in an abdominal lymph node, The two mammary tumours were not metastases. No hyperplastic lesions of the cervix were found in more than a hundred old female mice, untreated or receiving injections of the solvent used (sesame oil). It is probable that more cervical tumours would be observed if there were not an earlier development of mammary tumours, and the authors are now carrying out excision of the latter in order to lengthen life. Lawrence and Gardner (636) describe a lymphatic leukaemia, transmissible by cells, which arose in a mouse (strain A) receiving injections of oestradiol benzoate over a long period. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 545

Loeb, Suntzeff and Burns (657) describe certain changes in the generative organs of female mice (penetration of the uterine glands into or through the uterine muscle and metaplasia of the cylindrical to squamous epithelioma) which are found in mice under treatment with oestrogens, and occur, though much less often, in control mice, “ as a rule becoming more frequent with ad- vancing age. Under the influence of estrogen they are produced, on the whole, the more readily the greater the dosage used and the more continuous its action.” In a second paper of considerable length, which is illustrated by some good photomicrographs, Suntzeff, Burns, Moskop, and Loeb (809) record precancerous and carcinoma-like growth in the epithelium of the cervix and vagina in one out of 128 control mice, and in 25 out of 234 mice injected with oestrogens. These changes are gradual and represent a continuous series. “The study of the changes which take place in vagina and cervix with ad- vancing age and under the influence of long continued, experimentally produced stimulations by hormones, which ultimately may lead to the production of carcinoma-like proliferations, is not compatible with the view that somatic mutations in the stimulated tissues are the immediate cause of their cancerous transformation.” Bang (411) obtained metaplasia and down-growth of epithelium, with formation of cell nests, in the uterus of guinea-pigs receiving subcutaneous injections of oestrone benzoate. Korenchevsky and Hall (611) give illustrations of the squamous metaplasia in the uterus of the rat following the injection of oestrone (9 rat units daily for three or four weeks) ; this change is prevented by simultaneous large doses of progesterone. In a later note (613) the same workers report that, with the doses used, adenoma-like changes in the uterine glands and squamous metaplasia in the uterus were more pronounced in rats receiving oestrogens and androgens than in those receiving the former only. In rabbits receiving injections of folliculin (0.1 mg. twice weekly) Pierson (744) obtained squamous metaplasia and deeply infiltrative growth of the uterine epithelium with calcification and formation of cartilage and bone. The epithelial new growth penetrated the whole thickness of the uterine muscle, and resembled carcinoma very closely. The condition was similar in rabbits receiving in addition applications of tar to the ear. Pierson (745) describes also in a female rabbit, treated with folliculin as above for three years, a mamma-like neoplasm extending over almost the entire stomach, lying partly in plain muscle and partly in a layer of striped muscle on the surface of the stomach; there was nothing to indicate the origin of either of these tissues. There were also considerable squamous metaplasia and downgrowth of the gastric epithelium. Nelson (712) injected oestrone and oestradiol in various forms into guinea- pigs for periods from three weeks to fourteen months and obtained cystic and adenomatous hyperplasia of the uterine epithelium with metaplasia. ‘‘ This condition of metaplasia is most marked in the cervical regions where down- growths from the epithelium penetrate the submucosa and show pronounced keratinization and pearl formation.” Some of the animals showed also multiple subperitoneal fibromyomata. 546 J. W. COOK AND E. L. KENNAWAY In a long series of papers (648-656) Lipschutz and his fellow-workers have described fibromyomas of the uterus (subserous, myometrial, least often sub- mucous) and of all parts of the peritoneum in female guinea-pigs receiving either injections of oestrone, oestradiol and its esters, or tablets of oestradiol subcutaneously. These tumours, which may be invasive, are classed as benign, since they retrogress on cessation of treatment. (On the neoplastic effect of stilboestrol, see Lipschutz and Vargas: Lancet 1: 541, 1940.) Koref, Lipschiitz and Vargas (610) injected oestradiol benzoate in olive oil subcutaneously in male guinea-pigs and found very numerous small fibroid tumours (the microscopic appearances are not illustrated) on the peritoneum covering the abdominal wall, diaphragm, spleen and stomach, with more diffuse fibrosis around the spleen and kidney and in some parts of the mesentery. This type of growth occurred also in some females. In castrated males the changes were less pronounced and did not assume the type seen generally in females, in which the tumours were larger and less diffuse than in the male. Control injections of olive oil produced no such effects. The oestrogen had a considerable growth-checking action, the body weight for oestrogen-treated males averaging 538 gm. as compared with 805 gm. for animals injected with oil alone; the figures for females were 445 gm,and 726 gm. respectively. Jedlicky, Lipschutz, and Vargas (594) describe similar growths in either sex following injection of two other esters (caprylate and 17-benzoate-3-n- butyrate) of oestradiol. In castrated males bilateral tumours may appear at the sites of incision and ligature of the spermatic cord. Lipschutz and Iglesias (649), Lipschiitz, Vargas, and Iglesias (656), and Vargas and Lipschutz (826) gave subcutaneous injections of oestradiol benzoate in olive oil to 24 castrated female guinea-pigs, the total of the amounts injected three times weekly in the course of four months ranging from 1.2 to 4.0 mg. The animals showed (a) the characteristic hypertrophy of the mammary gland; (b) metrorrhagia in most cases; (c) retarded body growth. In all but two animals very numerous connective-tissue tumours were found in the wall of the uterus and also scattered over the abdominal cavity. Micro- scopically the structure was that of fibromyoma with a strong tendency to fibrosis. The tumours might penetrate smooth muscle, the striped muscle of the diaphragm and of the abdominal wall, or the pancreas and the liver, but did not penetrate the epithelium of the intestine or kidney. In some parts there appeared to be a proliferation of the endothelium of the lymphatic channels and of the peritoneal surface. In the third paper Vargas and Lipschutz show that the extragenital tumours (of the mesentery, abdominal wall, spleen, and surface of the stomach), which may be very abundant, may appear on the whole earlier than the uterine tumours. Moricard and Cauchoix (693) report the production of similar tumours in female guinea-pigs following the injection of oestradiol benzoate. Zuckerman and Morse have described cystic endometrial hyperplasia in a rhesus and a mangabey monkey, and in a chimpanzee (860, 859), following injection of oestrone, and squamous metaplasia of the cervix uteri in the rhesus monkey (858). McEuen (677) observed a number of malignant tumours in rats receiving CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 547 injections of oestrone, sometimes with application of other agents (tar, kiesel- guhr, silk threads) as well, but does not claim statistical proof for an action of oestrone in producing these tumours. He also (678) gives a fully illustrated account of the incidence of tumours in rats receiving oestrone, or testosterone, over long periods, together with applications of tar to the skin. Zondek (857) obtained pituitary tumours in 28 rats after administration of oestradiol benzoate in various ways, in some cases with simultaneous application of 3:4-benzpyrene to the skin. He discusses the efficacy of various solvents. Zondek (856) also describes experiments in which he subjected 240 in- fantile rats to biweekly administration of oestradiol benzoate parenterally or by application to the skin, 5000 units being the usual dose. All showed retarded growth and arrested sexual development. After four months the males showed enlargement of the pituitary, while in the females the gland remained grossly normal. After eight months pituitaries in both sexes were transformed into enormous tumours, which caused death in 29 of 35 rats. No tumour developed in less than seven months; the duration of treatment was found to be of more importance than the absolute amount of hormone given. Deanesly (492) found haemorrhagic pituitary tumours " which would probably eventually have proved fatal " in each of 3 rats in which oestradiol tablets (11.8 to 16.5 mg.) had been implanted subcutaneously for 270 days. Two of the tumours weighed 236 mg. and 356 mg. (normal pituitary 8 mg.). The histologic changes produced in the pituitary of the rat by oestrogens, apart from the development of tumours, are described. Wolfe and Wright (852) describe adenomatous enlargement of the pituitary, which may reach 15 to 20 times the normal weight, in rats receiving injections of oestrone. These adenomata are composed of chromophobe cells. The various forms of hyperplasia found in rats receiving oestrone are described and compared with those that occur spontaneously in' old female rats. Perry and Lockhead (743) gave repeated applications of oestrone to the skin of 131 female mice, of which some had received implantations of 1: 2 :5 :6- dibenzanthracene in the breast or uterus. The number in this latter group is not stated, nor is any mention made of the solvent for the oestrone. In 3 mice, of which 2 had received dibenzanthracene, tumours of the pituitary were present. No pituitary tumours were found in 97 controls. Korenchevsky and Hall (612) studied the effect of male and female hormones upon the hypophysis of the rat, with the results set forth in Table xxv. Bradbury (441) has published data on the oestrogenic activity of 9:lO- dipropyl-9 :10-dihydroxy-9 :10-dihydro-l :2 :5 :6-dibenzanthracene, its 5-methyl derivative, and a number of allied compounds. Supniewski and Hano (811) examined the pharmacological action of 9 :10- dihydroxy-9 : 10-di-n-propyl-9 :10-dihydro-l :2 :5 :6-dibenzanthracene upon the circulation and various organs. Oestrus lasting four to five days is produced in mice by 0.06 mg. McCluskie and Niven (675) found no oestrogenic action by the sarcoma- producing compound styryl 430, nor by the related compounds styryl 245, 548 J. W. COOK AND E. L. KENNAWAY

TABLEXXV: Efed of M& and Femule Hormones on Rat Hy@physis (Korenchcvsky and Hall)

Hormone injected (mg. per week) Average weight of Oestradiol hypophysis, female ____ Male hormone dipropionate rats, in mg. ..I None (control rats) None 11.9 None 0.09 98.0 None 0.2 132.2 Androsterone 7.5 None 10.8 Androsterone 7.5 0.09 48.0 Testosterone propionate 2.25 None 10.9 Testosterone propionate 2.25 0.2 60.2 styryl427, and anil421 (for formulae see original paper). The authors point out that the low solubility of these compounds may affect such tksts. Perry (741) produced oestrus in 3 out of 4 mice by subcutaneous injection of 31 mg. of 3:4-benzpyrene in lard (compare 27, 34). Two mice, of which one had not gone into oestrus, developed tumours at the site of.injection one month later. Freud (547) tested the oestrogenic activity of compounds applied per vaginam and sub cutem to rats. The threshold doses for intravaginal application were for oestradiol less than 0.03 y and for 9: lO~hydroxy-9:lOdipropyl- 9: lO-dihydro-l:2 :5 :6dibenzanthracene 12.5 y; for subcutaneous application the threshold doses were 0.125 and 50 y respectively. A dose of 10 mg. of 3 :4-benzpyrene-cycZo-penteno-phenanthrene applied intravaginally was in- effective. IV. Systemic Effects of Carcinogenic Compounds and of Tumours The separation of papers dealing with the local effects (Section 11) and the more remote effects (Section IV) of t4e carcinogenic hydrocarbons, which was adopted in the two previous reports, has been maintained here, although the great development of investigations upon the blood, lymphoid tissues, and reticulo-endothelial systems makes the distinction between the more and the less remote actions difficult to draw. Parsons (734, 735) examined the changes in lymph nodes of mice bearing various forms of primary induced, grafted, and spontaneous tumours, as well as of those under treatment with agents promoting (x-radiation) or inducing (carcinogenic hydrocarbolis) tumour development. In all, nine classes of mice were examined. The chief carcinogenic compound used was sodium 1:2 :5:6dibenzanthracene-9: lO-endo-&(cis) succinate (32) injected (0.3 C.C. of a 0.4 per cent watery solution) subcutaneously thrice weekly in 230 mice. One of these mice after 11 doses developed a spindle-cell sarcoma, which was excised on the twenty-second day and grew readily when grafted. This seems to be the shortest recorded time in which a sarcoma has been induced. During the first two months the nodes showed some diminution of lymphocytes, widening of sinuses, and proliferation of littoral cells; in the third and fourth months these changes became intensified; large reticulum cells appeared at the periphery and formed thick cords growing inwards into the medulla, and in these cells deposits giving the prussian blue reaction for iron were found. Cells resembling mast cells appeared in the vicinity of these ironcontaining CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 549 deposits. Similar changes occurred in rats treated in the same way and in mice injected with the following compounds : sodium 20-methylcholanthrene- 11 : 14 - endo - ap - succinate, sodium 1: 2 :5 :6 - dibenzanthracene - 9 : 10 - endo -up- (trans) succinate, 3 :4 :5 :6-dibenzcarbazole, and 3-methyl-1 :2-benzanthracene. When a sarcoma develops at the site of the injection of these compounds, all the changes in the nodes described above are accentuated. The lymphocytes may be reduced to small groups, the sinuses enlarge and contain macro- phages bearing iron-containing granules, and the fixed reticulum cells prolif- erate. The shift in the proportion of lymphocytes in the blood during the induction of tumours (301,464) may perhaps be correlated with these changes in the lymphoid tissue. Halberstaedter (5 73) describes changes in the lymph nodes, resembling those recorded by Parsons (734, 735)), in sarcoma-bearing mice and hamsters bearing grafted sarcomas induced originally by 3 :4-benzpyrene. He empha- sises (a) the high proportion, about 70 per cent, of animals showing metastases, which were not found in rats bearing similar tumours, and (b) the large number of lymph nodes involved. In all the hamsters showing metastases there were deposits in the lymph nodes. The process involved the axillary, inguinal, mediastinal and retroperitoneal nodes. ‘(We are here, therefore, dealing with a dissemination resembling a systemic disease. . . . In some cases it is possible to transmit tumours by the subcutaneous injection of blood from hamsters with metastases.” (See also Halberstaedter : Am. J. Cancer 38: 351, 1940.) Judina (5986) describes reticulo-endotheliosis and myeloid proliferation in mice after injections of methylcholanthrene. Mider and Morton (684) applied methylcholanthrene (0.5 per cent in benzene) to the skin of 60 dilute brown mice twice weekly, by the method of Murphy and Sturm, the application being made to nine different areas of the skin in succession, to avoid the production of local tumours. This effort to avert the carcinogenic action of methylcholanthrene was not very successful, 89 21 epitheliomata of the skin were produced. “ Of the 60 animals with which the experiment was commenced, 48 developed one or more malignant neoplasms. Ten died without tumours between the 26th and the 169th day. Two were lost.” Forty mice developed leukoses (see Table XXVI). Between 80 and 90 per cent of the breeding females of the dilute brown strain develop breast cancer at a mean age of 10.6 months, while the incidence of this form of cancer in virgin females is 51 per cent. Lung tumours occur in less than 5 per cent. According to a personal communication from Dr. C. C. Little to the authors, lymphoblastoma occurs commonly in both sexes between 650 and 800 days, the abdominal lymph nodes being most frequently involved. Thus, in the untreated mice mammary cancer appears before lymphoma. Under the action of methylcholanthrene this order is reversed, and the mice develop lymphoma in less than one half the time required for the spontaneous disease to appear. (Cf. Brues and Marble: Am. J. Cancer 37: 45, 1939.) Dobrovolskaya-Zavadskai’a and Rouyer (506) injected “ a very weak dose ” (amount and solvent not stated) of 1 :2 :5 :6-dibenzanthracene subcutaneously in a strain (XXX) of mice which in eight years had shown no spontaneous mammary carcinoma or lymphadenomatosis. In 102 mice which 5 SO J. W. COOK AND E. L. KENNAWAY

TABLEXXVI : Occurrence of Lcukoscs in Dilute Brown Mice Trealed with Mdhylcholanthrene (Mider and Morton)

Number affected Lymph Hiatolodc Type of lymphoma "dy nodes characten of Other change8 - experiment affected lymphoma F. - General 12 69-204 Cervical, axil- Same as thoae of Infiltration of liver lymphornatosis lary, inguinal, spontaneous disease and of many other up to 1 cm. in Large atypical cells organs:spleen very diameter: peri- spreading through large: high lymph- toneal and me- capsule and infil- ocyte count in diastinal less trating surrounding blood of some ani- enlarged tissues mals

Mediastinal 2 Not statec Mediastinal Same as above Spleen not en- lymphoma only larged: normal blood count

Nonmalignant 1 Not statec None Not described nor Infiltration of extra-medullar illustrated spleen, liver and myelopoiesis marrow: rpleen very large: hyperplastic bone marrow

Reticulo-endo- 1 130 t hcliosis ._____ - lived for more than three months they obtained 19 sarcomas at the site of injection (one of these animals showing leukaemia, also), 2 cases of generalized lymphadenomatosis, and one lymphosarcoma of the thymus. Csato, Wetzler-Ligeti, and Wiesner (486a) injected 1: 2 :5 :6dibenzanthra- cene (1 or 2 C.C. 0.3 per cent in olive oil or benzene) subcutaneously in 20 rabbits at intervals of ten to twenty days for periods up to thirteen months, the total dosage of hydrocarbon being of the order of 70 mg., which is near the maximum amount tolerated. The animals became cachectic but recovered when the injections were suspended. In the majority of animals the hydrocarbon caused a fall in the Congo Red index (indicating a reduction in the activity of the reticulo-endothelial system), which was not produced by the solvents alone, but in several instances considerable fluctuations occurred. In some rabbits prolonged administration of the carcinogenic compound was required to produce indices as low as those observed after one or two doses of a strongly negative-restropic pituitary extract. The possibility is suggested that the hydrocarbon inhibits the secretion of positive restropin by the pituitary. Other changes in the rabbits receiving the hydrocarbon were haemorrhagic graafian follicles and atrophic changes in the thyroid. No tumours developed during the period of the experiment. A subsequent paper by the same authors (4866) describes experiments in. which extracts of blood (5 c.c.) taken from rabbits receiving injections of 1: 2 :5 :6dibenzanthracene in benzene or olive oil, or of these solvents alone, were injected into other rabbits and the effect in them upon the absorption of Congo Red was noted. The effect in the second rabbit was in most cases a fall in the activity of the reticulo-endothelial system as indicated by the rate of disappearance of the dye. The changes in CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 551 the rabbits receiving the carcinogenic hydrocarbon were in most cases con- firmatory of the results described in the previous paper (486a), i.e. there was a decrease in the absorption of dye. It is noteworthy that the blood of one rabbit, in which an increase instead of a decrease occurred, produced in the second rabbit an increase instead of the usual decrease of absorption. Two controls only are described; the blood of one rabbit receiving benzene and of one receiving oil only produced increased absorption in another rabbit. Clarkson, Mayneord, and Parsons (464), continuing the observations of Mayneord and Parsons (301), found that general x-irradiation (a single dose of 550r) of mice (dilute brown) increased the number and rapidity of development of sarcomas induced by subcutaneous injection of sodium 1: 2 :5 :6- dibenzanthracene-9 : 10-endoup-succinate. The tumours of the irradiated animals were denser and gave a lower percentage of positive grafts (50 to 80 per cent) than those of the non-irradiated animals (100 per cent). A large number of data are given for the effect of x-radiation upon the blood, lymphoid tissue, and spleen in mice and rats. Storti and Storti (806) injected 3:4-benzpyrene (1 per cent in olive oil) into the right and left femur alternately of 100 rats, eight times in eight weeks (total: 8 mg. hydrocarbon). Of 75 rats which lived longer than two months, 12 showed noteworthy changes in the haemopoietic system after seven to ten months. (1) Anaemia, appearing after eight months, mostly of the hyperchromic type. The red cell count fell in some instances from the normal 7 or 8 million to 1 million or less. In 4 rats there was considerable erythroblastosis, i.e. normoblasts, perhaps twice as many as other nucleated cells, which numbered 100,000 to 150,000, but this condition was not proportional to the anaemia. Red cells containing Jolly bodies were increased. (2) Leucocytosis: 30,000 to 160,000 white cells (normal lS,OOO), with a reversal of the ratio of granulocytes to lymphocytes (neutrophils 79 to 93, lymphocytes 3 to 13 per cent). There were observed, also, polymorphs of abnormally large size; a small percentage of myelocytes, myeloblasts and metamyelocytes ; 0.33 per cent large non-granular cells of haemocytoblast type. (3) The marrow and spleen were affected in all 12 rats. Fat disappeared from the marrow, in which cells of either the erythroblastic or myeloblastic type predominated in different animals. Apparently the marrow in all parts of the body was thus affected, but the authors’ statement on this important point is not clear. The spleen weighed from 1.5 to 4 gm. (normal 0.3 to 0.7 gm.), with extensive myeloid metaplasia. In some cases there was considerable megakaryocytosis. No myeloid change was observed in the liver. The lymph nodes were not affected. The authors discuss various possible ways in which these changes may have come about. Tchakhotine and Kobozieff (8 16) examined the blood of mice (“ blanches ordinaires ”) receiving 3 :4-benzpyrene either by subcutaneous injection in ‘( oil ” or in benzene (the latter not a usual solvent for injection) or by application to the skin in benzene. Each injection was followed by a diminution of the total leucocyte count, e.g. to one-half, with restoration to the normal level within twenty-four hours, the fall being due to a loss of lymphocytes. Subse- quently nothing abnormal was observed until the tumour appeared, when an 552 . J. W. COOK AND E. L. KENNAWAY increase in total leucocytes and in the proportion of polymorphs occurred. The effect of application to the skin was quite different; the total count rose during the period of tumour induction (e.g. from 12,000 to 46,000), due to am increase in polymorphs, whereas in the unusual cases in which painting failed to produce a tumour the blood showed only a slight increase in lymphocytes, which did not reach the point of a true leucocytosis. The development of spontaneous tumours in mice, or in man, is not accompanied by any such changes in the leucocytes, as was pointed out by Tchakhotine in an earlier paper. In that paper (815) Tchakhotine described the changes in the leucocytes of mice accompanying the growth of three kinds of tumours, namely (a) spontaneous mammary carcinoma, in strain RIII, (b) grafted sarcoma 37S, (c) cancers produced by 3:4-benzpyrene applied to the skin or injected under it. (a) RIII has normally a high leucocyte count (20,000 to 24,000) and no change was seen before the development of spontaneous turnours, which appear generally at the end of the first year, nor until they had reached the size of ('a large walnut," when the polymorphs increased. The author remarks in this connection that spontaneous tumours in man are not accompanied by leucocytosis. (b) Animals grafted with sarcoma 37s showed a progressive increase in polymorphs, beginning while the graft was still quite small. (c) The growth of tumours produced by 3:4-benzpyrene was accompanied by an increase in polymorphs, and the total leucocyte count reached 60,000 or more. Tumours caused by 1 :2 :5 :6-dibenzanthracene appeared to produce a still greater leucocytosis.

Lanza (631) injected 3:4-benzpyrene (1 per cent (( oily " solution; 12 to 21 mg. in four to seven months) into the bone marrow of the two femurs, alternately, of 70 rats and in 10 of these observed " the appearance of the typical process of myelogenous leukaemia." There was leucocytosis (41 ,OOO to 78,0OO), with complete inversion of the lymphocyte-granulocyte ratio, and many immature cells, myeloblasts, myelocytes and metamyelocytes, and un- differentiated cells of the nature of haemocytoblasts and reticulo-endothelial cells often showing phagocytosis. In the marrow-f what bones is not stated-there was complete replacement of fat by myeloid cells, and the spleen showed myeloid metaplasia and was five to eight times the normal size. Only one of the 10 rats developed a sarcoma at the site of injection; inoculation of blood or spleen emulsion from them induced leucocytosis of immature granulocytes, with haemocytoblasts, and myelogenous metaplasia in the spleen and lung. Of the rats which were not considered to have developed leukaemia, 18 showed changes of the same type (leucocytosis of immature granulocytes), and 20 showed anaemia and erythropoiesis in the marrow. In both these groups there was great enlargement of the spleen with myeloid metaplasia. (Cf. Mayneord and Parsons, 301.) Judina (597) injected 0.1-0.2 C.C. of a 0.5 per cent solution of 1:2:5:6- dibenzanthracene in chicken fat every six days for five months subcutaneously in 45 chickens, and observed '' one case of leucaemia, 3 cases of aleumdc myelosis including one attended by sarcoma, 3 cases of erythroblastosis and CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 553 1 case of sarcoma without leucotic manifestations.” Some of the birds were observed over an eight-month period. Judina (5984 also injected 1: 2 :5 :6-dibenzanthracene (9 mg.) in mice (number not stated) and found at the end of four and a half months fusiform sarcoma accompanied by myeloid leukaemia. The number of leucocytes rose from 8,000 or 10,000 to 126,000 (large mononuclears 29 per cent, myelocytes 20 per cent). The spleen showed myeloid change and numerous giant cells. The tumours accompanied by leukaemia were grafted and produced the same changes, but the tumour sometimes occurred without leukaemia and con- versely. When a tumour was formed without changes in the blood, there was a proliferation of myelocytes in the liver and the spleen. De Gennaro and di Grazia (553) applied 3:4-benzpyrene (1 per cent in benzene) to the lumbo-sacral region of 15 mice, and in two of these observed after the development of an epithelioma the appearance of a lymphoid and a myeloid leukaemia respectively. The cellular changes in the blood and tissues are described in detail. Uher (824) injected bile subcutaneously and into the bone marrow in mice, rats, and rabbits, and obtained polymorph leucocytosis, myeloid foci in the liver, lymph nodes and spleen, and increase of myeloid tissue in the bone marrow, but no true leukaemia (cf. Burger and Uiker: Klin. Wchnschr. 16: 334,1937). Addition of 3:4-benzpyrene to the injected bile did not alter the results. Noble (721, 722) found that pellets of 10 mg. of crystals of methylcholanthrene, 1: 2 :5 :6dibenzanthracene, or 3 :4-benzpyrene, implanted subcutaneously in male rats, which were killed four weeks later, had no definite effect on body growth or upon the weight of the endocrine organs or gonads. ‘‘ Since these substances have a low solubility, it is possible that the amount absorbed was not great enough to produce any effect.” A dose of 50 mg. of 3:4- benzpyrene or 1: 2 :5 :6dibenzanthracene had a slight effect, the final average weight being 131 gm. with the former or 139 gm. with the latter compound, as against 159 gm. for rats receiving phenanthrene. In rabbits receiving 3:4-benzpyrene (20 doses of 1.8 mg./kg. in cholestenone-sulphonic acid solution, i.e., 20 doses of 1/10 of the mean lethal dose) Gummel and Rarei (565) found emaciation, from 2.5 kg. to 1.5 kg. before death in four to six months, without any sign of disturbance of the stomach. Cholestenone-sulphonic acid is haemolytic in dilutions of 1 in 10,000. These same observers (755) found that doses of 1 to 10 y 3:4-benzpyrene- cholestenone-sulphonic acid accelerated the germination and growth of green plants, which action was not due to the sulphonic acid. They also gave cholestenone-sulphonic acid and its compound with 3 :4-benzpyrene by mouth to young growing rats. The rats receiving the hydrocarbon showed an inhibition of growth which was not shown by those receiving the sulphonic acid alone. Cornil, Paillas and Castueil (478) describe the histologic appearances of the liver and kidney in two pairs of rats which had received a single subcutaneous injection of 1: 2 :5 :6-dibenzanthracene and 3 :4-benzpyrene, respectively. They observed multiplication and enlargement of the Kuppfer cells, and periportal infiltration. [In view of the variety of appearances found in the livers of rats, one might expect a larger series of animals to be examined,] 5 54 J. W. COOK AND E. L. KENNAWAY

Dietrich (495) found that after excision of a small portion of the skin of a mouse regeneration was not affected, in comparison with controls, by cutaneous application of 3 :4-benzpyrene at some distance from the area excised, from which it is concluded that there was no systemic effect upon regeneration. The changes produced at the site of application were loss of hair and thickening and downgrowth of epithelium (cf. Orr 728). The regenerative proc- esses after excision of the painted area appeared to be as rapid as at the distant spot. Wolfe and Bryan (850) injected hydrocarbons (5 mg. in 0.1 C.C. sesame oil) subcutaneously daily in pregnant rats, beginning on the first day of ges- tation, while controls, which showed none of the changes described below, received no injections, or the oil alone. 1:2 :5 :6-Dibenzanthracene was given thus to 38, and 3:4-benzpyrene to 17, rats. Vaginal bleeding occurred between the eighth and twelfth days, but the foetuses and placentas of animals killed on the eleventh day appeared normal. By the twelfth and thirteenth day, extensive haemorrhage from the margin of the placenta into the uterine lumen was found in practically all rats. Complete serial sections were made of 31 foetal sites. At the tenth or eleventh day the development of the foetus and of the placenta seemed normal, except that there were considerable amounts of blood in the uterine cavity at the junction of the decidua reflexa and decidua basalis. On the fourteenth and fifteenth days this condition was much more advanced, although there was considerable variation among indi- viduals, and among the foetal sites in a single rat. “ In two animals killed on the seventeenth and eighteenth days, respectively, the foetal and placental elements had disappeared completely from the uterus.” In two rats only the action of the slightly carcinogenic compound, 1:2- benzanthracene, was tested, with an identical result. “ Examination of the foetal sites on the fourteenth day revealed intraplacental hemorrhage, bleeding into the uterine lumen, and destruction of the foetal tissues.” The effects described above cannot be produced by injections begun in the later stages of pregnancy. Thus, four rats received 5 mg. of 1:2:5:6- dibenzanthracene on the fifteenth, seventeenth, nineteenth, and twenty-second days of pregnancy; all delivered normal litters and raised them successfully, but some amount of uterine haemorrhage and resorption of foetuses occurred in subsequent pregnancies, which was perhaps due to continued absorption from the site of injection. Hain (572) found that injection of 100 mg. of 3:4-benzpyrene into rats on the twentieth or twenty-first day of pregnancy had (‘no effect on the morphological development of the female foetuses ” and did not modify the effects of oestrone benzoate upon the foetuses. Rondoni and Beltrami (775) grafted sarcomata of the 2nd to 6th generation of tumours, induced in rats by 3:4-benzpyrene, into other rats, on the side opposite the site of a primary tumour induced in the same animal by 3:4-benzpyrene or methylcholanthrene. No resistance to the growth of the graft was shown by the rats bearing primary tumours, in comparison with controls. Druckrey, Hamperl, Herken, and Rarei (510) found that excision of the Flexner-Jobling carcinoma from rats was followed in 70 per cent of cases CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 555 by an abundant development of metastases, which was never seen in animals not operated upon. Hence the excision appears to have, or to remove, a systemic effect. Similar treatment of rats bearing the Jensen sarcoma was followed by metastasis in only 2 per cent. The authors then produced primary sarcomas in rats by a single subcutaneous injection of 3:4-benzpyrene (10 mg. in (‘oil ”); they had never seen metastases in over 300 rats so treated. The primary tumour was then excised; recurrence was very frequent, which is to be attributed to the highly infiltrative character of these tumours. Only one rat out of 40 was finally freed of tumour growth by surgical means. All of 7 rats which survived the excisions developed very numerous metastases, chiefly in the abdominal cavity. No evidence could be obtained that excision of the spleen, gonads or adrenals, or blockage of the reticulo-endothelial system, affected the resistance of rats to the two grafted tumours; whether any such experiments were made with the benzpyrene tumour is not clear. A similar development of metastases, after excision of a grafted Brown-Pearce tumour, is described by Solovieff (Abstract No. 1325, Index Analyticus Can- cerologiae, vol. 13, 1939). Dmochowski (498) immunised rats and rabbits with saline extract of normal tissues of rats and of tumours produced in rats by subcutaneous injection of 3:4-benzpyrene in lard, and found that the sera of the immunised animals gave complement fixation with heated extracts even more strongly than with homologous antigen. These cross reactions might be explained by the presence of normal antigens in tumour tissue. Heiman (579) in the course of an extensive study by Rasmussen’s method of the anterior pituitary in tumour-bearing rats examined some (‘with induced subcutaneous benzpyrene tumours and with induced rat sarcoma ” and found a moderate increase in chromophobe cells and the appearance of large degranulated basophil cells (3-6 per cent). Rats of either sex fed for over one year with 4’-amino-2 :3’-azotoluene showed a marked increase in chromo- phobes, a negligible number of basophils (0.5 per cent), reduced eosinophils (0.9 per cent), and an increase in degranulated cells. Aoki (394) found a diminution in the cholesterol content, especially of the adrenals, and also of the liver, spleen, heart and muscles, of rats grafted with hepatoma. A decrease of phosphatide was also observed in a number of the organs. Hashida (577) states that 4’-amino-2 :3’-azotoluene and butter yellow have a checking action upon the growth of embryo rat tissue grafted in the tissues of adult rats. Supniewski (810) examined the action of 1: 2 :5 :6-dibenzanthracene upon cardiac and unstriped muscle in the frog and rabbit.

V. Minimum Amount of a Compound, and Mode of Administration, for Carcinogenesis ,Mider and Morton (683) applied methylcholanthrene (0.5 per cent in benzene) once only to 44 C57 brown mice aged four to five weeks. The solution “ was applied to the back, from the occiput to the lumbar region, with two strokes of a No. 8 camel’s hair brush.” Epilation occurred within ten 556 J. W. COOK AND E. L. KENNAWAY days (cf. Orr, 728). Between the thirty-first and fortyeighth days, 16 mice developed 22 papillomas in the painted area; of these tumours, 15 regressed, 3 were found to be malignant (i.e. invasion of muscle) on the 228th, 124th and 114th days, and 4 remained stationary. If the volume of fluid thus applied were 0.1 C.C. the amount of hydrocarbon would be 0.5 mg. DobrovolskaYa-ZavadskaYa (501) administered 1: 2 :5 :6dibenzanthracene subcutaneously to mice in olive oil in amounts ranging from 0.1 mg. to 0.0012 mg. (1.2 y) and found that single doses of from 10 to 5 y frequently gave tumours at the site of injection, while even 2.5 y could produce sarcoma. “ With a dose of 1.2 y,” she concludes, “ we have probably reached the limit of the carcinogenic potentiality of dibenzanthracene.” Oberling, GuCrin, and GuCrin (723) injected subcutaneously in rats a range of doses of 3:4-benzpyrene from 0.05 to 75.0 mg. dissolved in olive oil, lard, or rat fat. With increasing doses there was: (a) a progressive increase in the percentage incidence of tumors from 14.3 per cent to 100 per cent; (6) a shortening in the latent period from nineteen to five months; (c) an increase in the incidence of metastases from 4.7 per cent with a dose of 1.0 mg. (lower doses, of 0.5, 0.1, and 0.05 mg., produced no metastases) to 50 per cent with 75.0 mg. These metastases were all in lymph nodes, except that of 3 animals receiving 75.0 mg., 2 showed pulmonary metastases, which had not been seen in 250 rats bearing tumours produced by 3 :Cbenzpytene. Rat fat as a solvent showed no adverse action upon the incidence of tumours, which reached 100 per cent with a dose of 25.0 mg. Stewart (804) implanted pellets composed of 5 per cent methylcholanthrene (0.6 to 1.0 mg.) and 95 per cent cholesterol subcutaneously in the abdominal wall of female C8H mice, which do not show sarcomata in this region either spontaneously or after implantation of cholesterol alone. At weekly intervals some of these mice were killed; some of the tissue surrounding the pellet was excised and implanted into other CaH mice and sections were cut of the pellet and remainder of the adjacent tissue. The histologic appearances around the pellets are described in full detail. The earliest transplant which produced a sarcoma was made on the forty-second day (the author points out that the presence of methylcholanthrene in the transplant cannot be wholly excluded) and the earliest appearance of progressive growth in a transplant was on the seventh day after implantation. Shear and Lorenz (796) give details of the technique and results of the use of hydrocarbon-cholesterol pellets in experiments on carcinogenesis. Andervont (382) injected two doses each of 0.8 mg. 1:2 :5 :bdibenzanthracene in 0.2 C.C. lard subcutaneously in mice (2nd generation of hybrids of strains A and C57) and six months later transferred some of the material in amounts of 0.05 to 0.1 C.C. from mice which had not developed tumours to 9 CaH mice, of which 7 produced sarcomas at the site of injection. Woglom (847) passed threads impregnated with crystals of 3 :4-benzpyrene through the kidney, liver, testis, or uterus of rats, while another piece of the thread was passed through the subcutaneous tissue as a control. Of 381 rats ,which lived for from 157 to 630 days, 29 showed “ progressively growing tu- mows at the site of the subcutaneous thread while no tumours were formed in the internal organs.” Of 16 subcutaneous tumours examined microscopi- CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 557 cally, 8 were myosarcomas (60), 1 a liposarcoma, 1 a fibrosarcoma, and 6 polymorphous or spindle-cell sarcomas. A similar experiment, of which no details are given, upon mice gave the same result, The author concludes that (‘relatively gross trauma does not lower the resistance of glandular epithelium to benzpyrene in the rat.” (Cf. the production of tumours of the lung by threads impregnated with 1:2 :5 :6- dibenzanthracene by Andervont and Lorenz, 202 .) Oberling, GuCrin, GuCrin and SanniC (725) compared the action of 3:4- benzpyrene injected subcutaneously in rats in olive oil, lard, and rat fat, and observed no distinct differences in the numbers of tumours produced nor in the occurrence of metastases. Morton and Mider (697) extracted fresh minced mouse carcasses, after removal of the stomach and intestines, with petrol ether and used the residue as a solvent for 3:4-benzpyrene; the yield of tumours was compared with that produced by the same hydrocarbon in two other media (Table XXVII). All surviving animals were killed thirty-two weeks after the injection.

’rADLE XXVII: Sarcomata in C57 Black Mice Receiving Single Injections of 0.25 fflg. 3 :4-Benspyrene in Inguinal Region (Morton and Mider)

Mice died or Medium Number of mice Total number of tumours killed without turnour8

Sesame oil 46 36 10 Colloidal in water 46 23 23 Mouse extract 44 1 43

On the use of homologous fat as a solvent in carcinogenesis, see also Chal- mers and Peacock (147) and Morelli and Dansi (690, in Section VI). VZ. Action of Carcinogenic Compounds on Tumours Haddow (570) observed the action upon sarcomata induced in rats or mice by 1: 2 :5 :6-dibenzanthracene, sodium 1 :2 :5 :6-dibenzanthracene-9 : 10- endo-ap-succinate, 3:4-benzpyrene, cholanthrene, and methylcholanthrene, of one or two intraperitoneal injections either of the compound use for induction or of others ( 1: 2 :5 :6-dibenzanthracene, 1: 2 :5 :6-dibenzacridine, 3 :4- benzpyrene, methylcholanthrene, 6 :7-dimethyl-1 :2-benzanthracene, 10-methyl- 1:2-benzanthracene, 1 :2 :5 :6-dibenzphenazine, or styryl430). All these compounds had been shown previously to possess some degree of growth-inhibitory power when tested against spontaneous mouse tumours or transplanted rat tumours or both. These primary chemically induced sarcomata showed as a class considerably less susceptibility to inhibition by carcinogenic compounds than did the spontaneous and grafted tumours. This relative resistance was, however, not specific, since tumours induced by a given carcinogenic compound were not significantly more resistant to the inhibitory action of the same compound than to that of other carcinogenic substances. Haddow (569) and Haddow and Robinson (571) record the data on inhibition of tumour growth obtained with 96 compounds in 348 experiments, of which each involved either a mouse bearing a spontaneous cancer, or a group 558 J. W. COOK AND E. L. KENNAWAY

Number of experiment8 in which result WM Total Inhibition Weak inhibition No inhibition

34 carcinogenic compounds. . . . , . . . 180 34 nontarcinogenic compounds.. . . 86

Thus, omitting results classed as weak inhibition, 86.5 per cent of 171 experiments with carcinogenic substances showed inhibition, while 79.7 per cent of 79 experiments with nontarcinogenic compounds gave no inhibition. The association is undoubted and extremely significant (x2 circa 100). It is pointed out that some carcinogenic compounds did not show inhibition (e.g. S-ethyl-1: 2-benzanthracene, 2’-methyl-, 9 :lOdi-n-butyl- and 9 :10-dibenzyl- 1: 2 :5 :6dibenzanthracene, phenanthra-acenaphthene) . The weakly carcinogenic compound 3 :4-benzphenanthrene, which is of special importance on account of its divergence in structure from 1: 2-benzanthracene, was found to have no inhibitory action on the growth of the Crocker sarcoma 180, while its actively carcinogenic 2-methyl derivative proved inhibitory both to that tumour and to spontaneous mammary cancer in the mouse. The compound 4’-amino-2 :3’ azotoluene, carcinogenic to liver cells, was also non-inhibitory. On the other hand, some nontarcinogenic compounds (4’-hydroxy- and 4’-methoxy-3 :4-benzpyrene, 2’:3’-naphtha-3 :4-pyrene) showed inhibition. Among the derivatives of 1:2-benzanthracene, there is a clear contrast between the lack of carcinogenic and of inhibitory power in those having one or two methyl groups attached to the angular ring, and the opposite condition shown by the 5 :6- and 6 :7-dimethyl-, 6 :’I-cyclo-penteno-, and 9-, lo-, and 9 :10- dimethyl-derivatives. (See also Acta, Unio internat. contra cancmm 3 : 342, 1938.) McJunkin and Wolavka (680) injected emulsions of 1: 2 :5 :6-dibenzanthracene in aqueous lecithin solution into the peritoneum of rats bearing subcutaneous grafts of a sarcoma arising originally in the uterus. “ Of fifty-one tumor-bearing rats that received injections of the dibenzanthracene-lecithin emulsion, the tumors completely disappeared in twenty. In many of the others the injections caused profound slowing of tumor growth, . , . Lecithin alone has little effect on the growth of this tumor.” Some of the rats died during the treatment. The authors offer evidence that the tumour in question was not very liable to regression, in that none of 29 rats that received intratumoral injections of 1: 2 :5 :6dibenzanthracene-lecithin emulsion showed complete regression and no tumours were caused to regress completely by intratumoral injections of methyl-thionine-chloride, sodium oleate and cysteine hydrochloride, which had in the hands of others caused malignant growths to regress and disappear. Morelli and Dansi (690) compared the inhibitory effects upon the Walker rat carcinoma of subcutaneous injections of 1: 2 :5 :6dbenzanthracene and CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 5 59

3:4-benzpyrene dissolved (a) in lard, and (b) in rat fat, while controls received lard only, and also the inhibitory effects of 3 :4-benzpyrene injected subcutaneously in lard and intravenously in colloidal suspension. “ The results proved that the highest inhibitory power was obtained by subcutaneous injection of the hydrocarbons in lard. The colloidal solution given intravenously showed a less constant activity. In mixtures of homologous lipoids and fats (that is, fats obtained by extraction from rats and injected into animals of the same species) there is a total suppression of the inhibitory power for 3:4-benzpyrene and a distinct diminution for I: 2:5:6-dibenzanthracene.” These experiments were suggested by the observations of Peacock (147) that the carcinogenic action upon the fowl of 1 :2 :5 :6-dibenzanthracene was lost when the compound was dissolved in fowl fat. (See other abstracts and references in Section V.) Peacock and Beck (738) endeavoured to apply Haddow’s discovery of the inhibitory effect of carcinogenic hydrocarbons by using intravenous injections, whereby the risk of initiating a fresh tumour would be lessened. Mice were inoculated subcutaneously with squamous carcinoma 2 146 and divided into three series which received (a) intravenously 0.03 per cent colloidal 3 :4-benzpyrene, (b) intravenously 0.06 per cent colloidal anthracene, (c) no injections, while a fourth series, not inoculated with tumour, received similar injections of 3 :4-benzpyrene. Complete or partial regression of the tumour occurred in 6 out of 13 mice receiving 3 :4-benzpyrene (4 complete and 2 partial regressions) : ‘‘ Only the regressions of well defined tumours are recorded; regression began about fourteen days after tumour inoculations. . . .” Two of the mice in which tumours regressed were re-injected, and found to be immune to the tumour. No regressions occurred in 9 controls receiving anthracene, but regression of a tumor 2 mm. in diameter occurred in another mouse receiving anthracene. The tumour used gives 70 per cent of progressively growing tumours. “ Two out of 120 controls in this experiment resulted in small palpable nodules which disappeared within about ten days.” The method has been tested in one human patient, with a rapidly growing and fatal sarcoma of the nose, who received 31 injections of 6 mg. 3:4-benzpyrene each, in thirty-nine days, without any effects, injurious or beneficial. No toxic effects were found in mice receiving daily injections of 0.3 mg., nor in rabbits or fowls receiving 0.5 mg. benzpyrene. Toxic effects apparently due to haemorrhage and haemolysis occurred in rats and goats. The authors suggest that intravenous injection is advantageous because a high concentration of hydrocarbon is reached, at any rate for a short time, while elimination by the liver is taking place, whereas the concentration resulting from subcutaneous or intraperitoneal injection is always low. The danger of initiation of new tumours was not eliminated, for one mouse receiving benzpyrene showed three adenomata of the lung, whereas no such tumours were found in more than 400 control mice. ‘‘ Fluoroscopic examination of the lungs of mice shortly after intravenous injections of benzpyrene colloid revealed fluorescent areas corresponding with deposits of benzpyrene in the capillaries. These may persist and may be responsible for the induction of such multiple adenomata.” 560 J. W. COOK AND E. L. KENNAWAY Bauer, Rarei and Gummel (416) state that all the seven patients with suitably situated cancers of the skin which were previously reported as treated with 3 :+benzpyrene (2 10) have remained cured for a period amounting in sum to four and a half years, and that no tumours were produced by the hydrocarbon. An inoperable mammary carcinoma in a man, with numerous lymph node metastases, received an intratumoral injection of 10 C.C. of a 0.5 per cent solution in ether of methylcholanthrene. The tumour sloughed off and left a granulating surface which showed no cancer in three portions removed for microscopic examination; no effect upon the metastases was observed. For this investigation Windaus prepared a series of water-soluble salts of benzpyrene, but tests of these by Domagk and the authors “showed that on the slightest alteration of the molecule the carcinogenic and with it also the anti- blastic action of the substance disappeared.” A 1 per cent watery solution of 3 :4-benzpyrene can be obtained by the use of sodium-cholestenone- sulphonate, but the latter is strongly haemolytic even in a strength of 0.01 per cent, and hence cannot be used for intravenous injection. Benzpyrene when given by the mouth is shown by fluorescence to remain in the intestinal con- tents and to be absent in the mucous membrane. The authors refer to a water- soluble benzanthracene preparation obtained by means of an organic acid, but give ng further information. Alapy (377) gave to mice and rats a preliminary treatment with very small amounts of 3 :4-benzpyrene, methylcholanthrene, and 1: 2 :5 :6-dibenzanthracene as a protection against tumours inoculated later. The neoplasms used were, in mice three highly virulent tumours-two carcinomata and a benzpyrene sarcoma-and in rats the Flexner-Jobling carcinoma. In mice the most effective dose was 1.5-3 y when the tumours were not inoculated until after forty to seventy days. The result was a weak protection in mice . . . retardation of the beginning of growth by five or six days, followed by slow growth for a further five or six days; then more rapid growth so that by the seventeenth or eighteenth day after inoculation the tumours were as big as those of the controls. Dibenzanthracene was rather more effective than the other two hydrocarbons. The rat tumour was not affected, but the body growth of young rats was retarded for months by even 30 or 40 y. No experiments with non-carcinogenic hydrocarbons are recorded and no figures for retardation of body growth are given. Lauber, Hildebrand and Schocke (634) in a paper of 24 pages describe in quite unnecessary detail the production of individual tumours in a number of mice by application of methylcholanthrene to the skin, and reach the not very surprising conclusion that injection into such tumours of a solution of methylcholanthrene in ether produces no more necrosis than might be due to the ether alone. On the basis of a single case of each kind, they suggest that very small amounts of methylcholanthrene, injected either into a tumour or at a distance from it, may accelerate its growth. Appel, Strauss, Kolischer and Necheles (395) found that the subcutaneous injection of 0.5 C.C. of a 0.1 per cent solution of 1:2 :5 :6-dibenzanthracene in benzene in rabbits for six weeks before the inoculation of the Brown-Pearce tumour, and thereafter until the death of the animal, led to (a) a greater number of “ takes ” (100 per cent as against 90 per cent), (a) a shortening CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 561 of the duration of life, (c) an increase in the dissemination, size, and number of the metastases, (d) the occurrence of metastases in organs (thyroid, supra- renal, spleen) which were not affected in the control animals. All the injected animals developed extensive carcinomatosis and none showed at any time any regression of the tumour, whereas the tumours in 25 per cent of the control animals regressed. In view of the small amount of hydrocarbon given (0.5 mg. twice weekly) control observations upon the effect of benzene alone would have been desirable. Bisceglie (434) states that epithelial tumours of the skin of the mouse induced by 3 :4-benzpyrene (1 per cent in benzene twice weekly) grow more rapidly if painting be stopped when the tumour appears. In this and four other papers (432, 435) this author gives a large amount of data on the production of tumours by 3 :4-benzpyrene. Boyland and Boyland (440) find that mouse tumours (sarcoma M.C.D.B.I. and Crocker NO), of which the growth is inhibited by administration of 1: 2 :5 :6-dibenzanthracene and 1 :2 :5 :6-dibenzfluorene, show values for glycol- ysis and respiration which are not significantly lower than the values given by untreated tumours, and this lack of effect is observed in the same tumours under the action of some other compounds (sodium sulphanilylsulphanilate, a-nitroso-&naphthol and 4 :4'-diamino-2 :2'-dinitrodiphenylmethane) which in- hibit the growth of these tumours. VII. Carcinogenic Compounds and Viruses Beard, Bryan, and Wyckoff (420) examined warts which were collected from about 160 wild cotton-tail rabbits trapped in Kansas and were sent to the laboratory in equal parts glycerine and 0.9 per cent sodium chloride solution at 5"-8". " Under these circumstances the virus activity of the growths seems to be maintained indefinitely." The material was ground with sand and subjected to preliminary and ultra-centrifugation (60,000 times gravity), of which details are given. Saline extracts remained infectious for several months at 5" to 8" and resisted heating at 67" for thirty minutes. Centrifuga- tion for one hour at 60,000 times gravity was found to remove over 99 per cent of the virus. Purified virus concentrates gave the usual test for protein, but a negative Molisch test. An analysis is shown in Table XXVIII.

Acetone- C N N+ S Ash solub!e H (Dumas) (Kjeldahl) Material _~~~~______~ Per cent dry 49.56 7.15 14.99 14.5 .944 2.2 2.5 3.2 weight - 562 J. W. COOK AND E. L. KENNAWAY After three or more ultracentrifugations the solution showed sharp and constant boundaries in photographs; if it were damaged in any way, the boundaries were less definite. The protein was most stable in the pH range 4.0-7.0. The isoelectric point was between pH 4.8 and 5.1; on the alkaline side of this region the protein bore a negative charge. The yield of protein varied from 1 mg. per gm. of highly infectious, naturally occurring warts, to 0.008 mg. per gm. of warts of low infectivity obtained by experimental inoculation. “ The yields of protein paralleled the virus activity of the various initial crude suspensions from which they were derived. . . . Despite extreme vari- ations in yield, the purified protein from different sources exhibited constant virus activity. . . .” This is illustrated by comparison of two batches of warts: one so-called poor batch, designated as 1354, and one good batch, i.e., of high infectiousness, designated as F. The good warts were from ten to a hundred times as active as the poor ones. “ There was, however, practically no difference in the disease-producing capacities of the virus proteins extracted from the two batches. Each consistently gave excellent papillomas when 0.1 C.C. of a solution containing lo-*grams of purified protein was used as inoculum. . . . The period of incubation was likewise related to the content of virus protein. Thus, for the crude extract of F the period was six to eight days; for 1354 it was fourteen days. The purified proteins, on the other hand, showed the same incubation periods on parallel dilution.” “An attempt also was made to demonstrate virus activity in domestic rabbit papillomas and to isolate heavy protein from them by differential ultra- centrifugation. For this purpose warts were obtained in domestic rabbits by inoculating large areas on the abdomen and side of the animals with crude saline extracts of cotton-tail rabbit warts or with purified virus protein. Ten experiments were made, 6 with glycerolated and 4 with kesh domestic rabbit growths.” Extraction and centrifugation were carried out as with the cotton-tail growths, and eventually solutions were obtained giving ‘‘ no, positive test for either protein or carbohydrate. In no instance was virus infectivity demon- strable in either the crude extracts or the ultracentrifugal fractions.” Beard and Wyckoff (419) continued their earlier experiments (212, 367- 369) on the pH limits within which the Shop papilloma virus remains active. They inoculated into the skin of susceptible rabbits initially active virus kept for various periods at different hydrogen-ion concentrations, while the molecular constitution of the protein in these solutions was determined by ultracentrifugal analyses. Glycerolated wart tissue known to be infectious was ground with sand, extracted with 10 parts of saline, cleared in an ordinary centrifuge, and then placed for one hour in a field of about 60,000 times gravity. The deposit was resuspended in saline and freed of colloidal material in a Swedish angle- centrifuge, and then ultracentrifuged as before. This process was repeated four or five times. By then the deposit was colourless and transparent and gave colourless, strongly opalescent solutions. By this process the heavy protein showing virus activity was freed from light tissue proteins and from various colloidal materials. Usually no chemical test for protein was given CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 563 by the supernatant liquid from the third or later sedimentations. The amount and homogeneity of the heavy protein from different batches of warts was variable. None was extracted from non-infectious tissue. Warts of low infectivity gave low yields with diffuse boundaries suggesting heterogeneity. “ Our very homogeneous preparations,” say the authors, “ have all been derived from highly infectious tissue.” For the present experiments 18.2 mg. purified protein were obtained from 105 gm. of wart tissue. Details of the buffer solutions and of the method of exposure of the virus to these are given. There was some loss of activity in all virus solutions kept for four weeks, but there was least loss in acid from neutrality to pH 4.2, while at pH 3.0 the inactivation was rapid and at pH 2.5 it was immediate. The activity was not long maintained in any alkaline solution and was lost instantly in solutions more alkaline than pH 10. In acid, addition of virus caused little change in the pH of a buffer, “ while on the alkaline side its pH was sharply lowered.” Details of the measurement of the rate of sedimentation by photography in ultraviolet light are given, and typical series of photographs are reproduced. The protein has its isoelectric point at pH 5. Between pH 4.2 and 5.8 it was too insoluble for measurement, while it dissolved on the acid side with the same sedimentation constant that it had at pH 6. This value was maintained with increasing acidity until near 2.8 the sedimentation rate dropped suddenly. “ This is undoubtedly due to a break in the protein molecule-light, ‘ unsedi- mentable ’ material could be seen in considerable amounts in the diagrams of very acid solutions.” In solutions more alkaline than pH 10 the protein was split into fragments that were smaller the higher the pH, as is the case with the tobacco mosaic virus protein. Both the acid and alkaline pH values at which there was molecular breakdown are, as closely as can be determined, those at which virus activity is lost. Between pH 7 and 10 solutions become non-infective at a rate increasing with the alkalinity, but this is not accompanied by a change in the sedimentation diagram, and hence this inactivation is due to chemical changes that do not alter the molecular size and shape. The purified protein shows two sedi- menting boundaries, of which the secondary one is the heavier. The nature of this character, which is common in heavier proteins, is discussed. Lauffer and Stanley (635) examined the stream double refraction of various virus proteins and found that this property was not shown by the elementary bodies of vaccinia and the Shope papilloma virus protein. Such negative results “ mean that the double refraction of the system is either zero or some value too small to be detected by the method employed. This may obtain with particles which are not sufficiently asymmetrical to be oriented appreciably in a viscous stream in which the shear is small. The elementary bodies, which are quite large, have been photographed and appear to be fairly symmetrical. Very small particles, on the other hand, regardless of the degree of asymmetry, can be oriented only by very strong mechanical fields because of the tendency to become randomized again owing to Brownian move- ment. Another possibility, however, exists which would account for low or unmeasurable values of stream double refraction. A consideration of Wiener’s theory reveals that in the case of isotropic asymmetrical particles, low or even 5 64 J. W. COOK AND E. L. KENNAWAY undetectable values of double refraction of flow will obtain if the index of refraction of the particles approaches that of the suspension medium. Failure to detect double refraction of flow in solutions or suspensions cannot be regarded as proof that the suspended particles or dissolved molecules are symmetrical." Mellanby (681) found that if a fowl bore both chemically induced (by tar or by 1:2:5:6-dibenzanthracene) and Rous tumours at the same time, the filterable agent of the latter is transmitted to and taken up by the former tumour, so that a cell-free filtrate of the chemically induced tumour may give rise to a Rous sarcoma, while an inoculation of cells from the chemically induced tumour will reproduce a tumour of this same type of cells. Generally the two types of tumour can be obtained from the chemically induced one only if a Rous sarcoma is present in the same bird, but in a few cases cell-free filtrates from the second grafted generation of dibenzanthracene tumours have given rise to Rous tumours propagable by filtrates, although no such tumour was present, while the cells from the grafted tumour gave rise to a tumour of the same type from which no active filtrate could be obtained. Injection of Rous filterable agent into a dibenzanthracene tumour does not cause it to regress, but may produce Rous sarcomatous tissue in it. Banting (412) injected 1 :2 :5 :6-dibenzanthracene (5 mg. in lard three times at fortnightly intervals) into 12 normal and 12 Rous-resistant fowls. Eight of the former and 6 of the latter series developed lesions having the histologic characters of dibenzanthracene tumours. " Thus it may be concluded that a high degree of resistance to Rous does not protect a bird agajnst the development of dibenzanthracene tumours." McIntosh and Selbie (679) found that injection of 1: 2 :5 :6-dibenzanthracene in lard into the pectoral muscle of fowls produced fibrous tumours in from sixty to ninety days, which were difficult to propagate. " On several occasions " they " obtained successful tumour passages of the Sheffield dibenzanthracene tumour (Mellanby: 11th Ann. Rep. Brit. Emp. Cancer Cam- paign, 1934, p. 81) by means of sand and paper-pulp filtrates," although they were never successful when a Berkefeld filter was used. Their experience has shown " that only filterable tumours can be successfully transmitted by means of sand and paper-pulp filtrates, and the difficulty of obtaining an active Berkefeld filtrate from this tumour is probably due to the larger size of the ViTuS." Ahlstrom and Andrewes (376) made intramuscular or subcutaneous injections of carcinogenic hydrocarbons in lard into rabbits, and five days later gave 3 to 5 C.C. of filtrate containing the fibroma virus of Shope intravenously. Observations were made of the localisation of virus at the site of injection of the hydrocarbon and of the occurrence of generalised fibromatosis. The results are summarised in Table XXIX. A second series of experiments, in which the virus was given subcutaneously or intradermally, was impaired by the poor condition of the rabbits, but the results appeared to show that the tumours were larger and more persistent in rabbits receiving benzpyrene, cholanthrene, methylcholanthrene, 5 :6-cyclupenteno-l: 2-benzanthracene, or chrysene, than in those injected with lard, phenanthrene, fluorene, or anthracene. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 565

TAnLE XXIX:Eftrt of Various Hydrocarbons on the Reaction of Rabbits lo the Infectious Fibroma Virus of Shope (Ahlstrdm and Andrewes) _._~___

Localization of virus to Hydrocarbon Site of injertion site of hydrocarbon Generalised fibromatonis inoculation -- 3 : 4-Renzpyrene Intramuscular + (persistent) - 6 - - Subcutaneous + (persistent) + (ear, legs, paws, jaw, spine, scapula) Intramuscular + (soon regressing) - 'I + (persistent) + (ear, jaw, legs) 'I + (soon regressing) - Subcutaneous + (soon regressing) Phenant hrene Intramuscular - Id It + (soon regressing) Id Subcutaneous + (soon regressing) Anthracene Intramuscular - - I, ,I - - ,' '' - - hlethylcholanthrene I + (persistent) f (rnr, forelirnhq, ribs) I' - - + (soon regressing) -

Amies and Carr (379) made a study of the des Ligneris (80, 81) fowl sarcoma which was produced in a culture of normal chick fibroblasts to which 1 :2 :5 :6-dibenzanthracene was added. The tumour-producing activity of cell- free extracts of this sarcoma can be removed by high-speed centrifugation, and the agent can be recovered quantitatively from the deposit. Cell-free extracts of the tumour produced progressive growth in ducklings and in this and other respects the tumour appears indistinguishable from the Fujinami sarcoma. Foulds and Dmochowski (544) found that the serum of a rabbit which had received injections of filtrate (sand and paper-pulp and Berkefeld) of a non-filterable dibenzanthracene fowl sarcoma neutralised filtrates of Rous sarcoma No. 1; the neutralising power was not abolished by absorption with normal fowl tissues. The particular antigen which elicits this reaction appears to be larger than the infective agent of Rous sarcoma. Dmochowski and Knox (499) immunised a rabbit with a 0.8 p membrane filtrate of the non-filterable tumour RFD2, a sarcoma induced in the fowl by 1 :2 :5 :6-dibenzanthracene, and found that the serum gave '' complement- fixation with active filtrates of Rous and Mill Hill 2 tumours and of fowl- and duck-grown Fujinami tumours. . . . Since RFD2 filtrates and heat-inactivated Rous filtrates also give a positive complement-fixation reaction, it is evident that both filterable and non-filterable tumours share a common antigenic factor." The authors conclude that '' the antigenic factor detected by complement-fixation is not predominantly species-specific." Woglom (848) injected ground, or frozen and thawed, tissue of mouse sarcoma 3 7 either subcutaneously or intraperitoneally into mice bearing " benzpyrene lesions " and found no acceleration of tumour growth such as might be expected if sarcoma 37 yielded an agent similar to the Shope papilloma or fibroma virus utilised in the experiments of Rous and Kidd (J. Exper. Med. 67: 399, 1938), Lacassagne and Nyka (286), Andrewes, Ahlstrom, 5 66 J. W. COOK AND E. L. KENNAWAY

Foulds and Gye (204), and Andrewes and Ahlstrom (J. Path. & Bact. 47: 87, 1938). VZZZ. Tumours of the Lung Andervont (385) injected 1:2:5:6dibenzanthracene into 296 C,H mice either subcutaneously in lard or serum or intravenously in serum, and found lung tumours in 99 out of 145 mice alive when the first of these tumours was noted (normal incidence 7 per cent after first year). Induced lung tumours begin to appear in CsH mice five or six months after subcutaneous injection of 1 mg. 1:2:5:6-dibenzanthracene, whereas in strain A such tumours appear in ten to twelve weeks. Hepatomas occur in the CsH strain in about 11 per cent of females and 22 per cent of males, and experiments did not show con- clusively whether this proportion could be increased by injection of 1:2 :5 :6- dibenzanthracene or methylcholanthrene. Andervont has published a series of four further papers on pulmonary tumours in mice. He found (386) that strain A mice, in which the lungs are more susceptible than the subcutaneous tissue to the carcinogenic action of 1:2 :5 :6-dibenzanthracene, developed pulmonary tumours within three months of subcutaneous injection of 0.8 mg. of the compound in lard. Tumours began to appear four weeks after injection, and by the eighth week “ virtually all the animals had lung nodules.” Similar results were obtained when the hydrocarbon was injected in horse serum, lard, olive oil or mouse-fat emulsion in water, or lard emulsion in serum. Amounts of 0.2 or 0.5 mg. of the hydrocarbon injected intravenously produced lung tumours in practically all the mice in six weeks, i.e., more rapidly than did 0.8 mg. subcutaneously. Similar results were obtained with methylcholanthrene. The second paper (387) records later results in the transplantation of pulmonary tumours of mice of strain A following parenteral introduction of 1 :2 :5 :6dibenzanthracene (200). Of 18 primary adenomatous lung tumours, 6 retained their original structure, 7 changed their type but remained epithelial, while 5 became spindle-cell tumours. Of the 5 tumours which later became spindle-cell, one arose spontaneously in strain A; the remaining 4 were induced in strain A, C, or CsH. The third paper (388) records the induction of lung twnours by subcutaneous or intravenous injection of 1 :2 :5 :6-dibenzanthracene in strains of mice (D, M, CS7 Brown, C57 Black, and D-C57 Black hybrids) which are resistant to the development of spontaneous pulmonary tumours. Andervont’s fourth paper (389) records the induction of pulmonary tumours in strain A by subcutaneous injection of 4’-amino-2 :3’-azotoluene. Seventy-eight per cent of the mice developed primary lung tumours, often multiple, between three and twelve months after the first injection. Experi- ments upon other strains appear to have given inconclusive results. The carcinogenic compound 3 :4 :5 :6dibenzcarbazole was injected subcutaneously in lard in strain A mice, of which 78 per cent developed from 1 to 4 pulmonary tumours each. This incidence is considerably higher than normal and the multiplicity indicates that the tumours were induced. Shimkin (800) gave methylcholanthrene or 1: 2 :5 :6dibenzanthracene (0.1 mg. in 0.1 C.C. horse serum and cholesterol, 202) by intratracheal injection to CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 5 67

TABLEXXX: Results of Inlratrached and Intravenous Injections of Methylcholanthrene (Shimkin)

Percentage of Average number of mice wlth lung lumg tumoura tumour8 per mouse Methylcholanthrenein horse serum Intratracheal 91 5.4 Intravenous 94 10.8 Horse Berum In tra t rachea I 30 1.3 Intravenous 10 1.o

Strain A mice, and compared the results with those of intravenous injections. mepercentage incidence of lung tumours was about the same in the two classes, but the intravenous method gave considerably more tumours per mouse (Table XXX). Two mice receiving methylcholanthrene, one intravenously and one intra- tracheally, " which are regarded as negative had lymphomatous infiltration throughout the lungs " (cf. Mider and Morton, 684). 1 :2 :5 :6-Dibenzanthra- cene was found to be less carcinogenic when adsorbed on charcoal (cf. 203). All the lung tumours were of the ordinary subpleural adenomatous type. Shabad (783) gives the following data for the incidence of lung tumours in a certain strain of mice,

Control mice more than eight months old ...... 8.0% Benzene applied to skin ...... 6.3% 1:2:5:6-Dibenzanthracene in benzene to the skin, all mice ...... 22.5% 1 :2:5:6-Dibenzanthracene in benzene to skin, mice surviving 6 months treatment 36.2% 1 :2 :5:6-Dibenzanthracene in sunflower oil, subcutaneously ...... 27.0-39.0% 3 :4 :8 :g-Dibenzpyrene in olive oil subcutaneously (giving 100 per cent sarcomas at site of injection) ...... 30.0% The tumours which developed under the influence of a carcinogenic agent were identical in structure with those occurring spontaneously. The incidence of these lung tumours could be raised from 8 to 25 per cent by applications of tar given so infrequently that they did not produce tumours of the skin (Cf. Kling et al., 604, 605). Grady and Stewart (563) injected 1: 2 :5 :6-dibenzanthracene or methylcholanthrene in lard, or lard alone, subcutaneously in strain A mice, and studied the lungs of 130 receiving carcinogens and 30 controls. The earliest pulmonary tumour was found on the thirty-second day in a mouse receiving methylcholanthrene. " From the fortieth day onward, tumours were found with increasing frequency ." All were adenomata resembling spontaneous lung tumours. No statement is made about the findings in the control mice. In fifty of these tumours which were grafted the appearance changed to that of spindle-cell sarcoma at the third or fourth generation (Cf, Andervont, 200). With regard to the sensitiveness of the lung as a test object for carcinogenic action, reference may be made to the work of Kling, Samssonow and Heros (604, 605), who painted 60 mice with " light oils " coming over at 75" to 200" from inclined retort coal tar diluted 1 in 10 with liquid paraftin. No skin tumours were produced. Thirty-seven mice died before any cancers appeared. Of the 23 remaining, 13 died of lung tumours, single or multiple, 568 J. W. COOK AND E. L. KENNAWAY sometimes filling the whole Iobe and showing the structure of alveolar epithelioma. The oil was subjected to chromatographic adsorption and a fraction redissolved in alcohol was precipitated with picric acid; adsorption and fluorescence spectra indicated that this material contained 3 :4-benzpyrene. On induced tumours of the lung, see also Peacock and Beck (738); Ander- vont (381, 383); Furth, Furth and Breedis (549).

IX. Tissue Culture and Cytology Earle and Voegtlin (512) examined the action of finely divided crystalline methylcholanthrene in amounts ranging from 0.2 mg. to 0.0002 mg. upon tissue cultures of fibroblasts of the rat (Wistar or Buffalo) and mouse (C,H). Both animals are highly susceptible to the carcinogenic action of this compound in vivo. After a latent period of some days there was a striking inhibition, leading to complete or nearly complete cessation of growth, and more or less severe cell destruction occurred in all cultures. The injurious effect was a function of concentration; thus complete degeneration was brought about by 0.2 mg. in twenty-eight days, and by 0.002 mg. in one hundred and thirty days, while an amount of 0.0002 mg. would almost certainly have proved lethal if the experiment had not been discontinued. In no instance was there any sign of stimulation of the cultures. In a series of cultures of mouse fibroblasts the shutting-off of all light corresponding to the adsorption spectrum of methylcholanthrene, down to 490 mp, did not alter the retardation of growth, but there was less cell degeneration. The fibroblasts exposed to methylcholanthrene did not acquire the morphological character of the cells of sarcomas induced in the same species by the same compound. A less complete experiment with 1 :2 :5 :6-dibenzanthracene gave confirma- tory results. (See also Earle and Voegtlin: Pub. Health Rep. 55: 303, 1940.) Larionow, Ivachentzova and Tchertkova (632) examined the action of 3:4- benzpyrene and 1: 2 :5 :6-dibenzanthracene on cultures of fibroblasts from the spleen. Benzpyrene (0.002 to 0.005 per cent) caused weak stimulation of growth for the first three to four days, and on the fourth or fifth day an arrest of growth and death. A concentration of 0.0005 to 0.001 per cent produced no stimulation, but death of the majority of cells after seven to twelve days. A concentration of 0.0001 per cent was without effect. Dibenzanthracene in saturated solution in the medium had no definite effect on the growth of fowl fibroblasts. Inoculation of such cultures into fowls gave negative results. Magath (659) observed a delayed (after three weeks) stimulating effect of carcinogenic hydrocarbons on the growth of cultures (skin and bits of tail) of mouse embryos. Attempts to produce tumours by inoculating such cultures into mice failed. W. H. Lewis (643) records " a peculiar type of mitosis characterised by extreme contortions, lobulations, and constrictions of the dividing cell and for a short period thereafter of the two daughter cells '' in cultures from a sarcoma obtained by subcutaneous injection of 1: 2 :5 :6-dibenzanthracene in a mouse and transplanted for over fifty generations. " The cells have remained practically the same since the first generation " and many show pinocytosis (296). CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 569

The explanation of these appearances and the mechanism of cell division in general are discussed by the same author in another paper (645), and in a third paper (646) the characters of cells from over 200 mouse sarcomas, of which most were produced by the injection of 1: 2 :5 :6-dibenzanthracene in the axilla of pure line mice, are described and illustrated. Bisceglie and di Grazia (433, 436) give data upon the growth in tissue culture of sarcomas produced by 3:4-benzpyrene, and upon the action of this compound upon tissue cultures exposed to light. Creech (484) added the choleic acids of 1 :2 :5 :6-dibenzanthracene (A ) , of phenanthrene (B), and of acenaphthene (C), or deoxycholic acid (D), to cultures of fibroblasts from embryonic mice (0.01 mg. per c.c.) and recorded the dimensions of outgrowth and mitotic counts, or the former alone. A caused an outgrowth 50 per cent greater than that of the untreated controls, while B and D had an inhibitory action. (‘The chromosomes showed a precocious splitting in the prophase in the cultures treated with 1 :2 :5 :6-dibenzanthracene choleic acid, 20-methylcholanthrene choleic acid, and methylcholanthrene in serum, but not in any of the four types of controls. Similarities to meiosis (reduction division) were occasionally found in cultures treated with 1 :2 :5 :6- dibenzanthracene and methylcholanthrene choleic acid where the metaphase chromosomes were lying closely in pairs, the members of which separated later to opposite poles, resulting in a reduction division, and in the ‘ node and loop ’ formation of the chromosomes.” Athias and Furtado Dias (398) describe the giant cells seen in sarcomas induced by methylcholanthrene, and the forms of mitosis occurring in them, and discuss the nature of these cells, which they do not regard as products of degeneration. See also Dias (494). Changes (vacuolation, fatty degeneration, nuclear fragmentation, coagula- tion of chromatin) produced by carcinogenic hydrocarbons in cultures of fibroblasts from dog embryos are described by Mauer (673). von Moellendorff (688) observed the effects (disturbance of the equatorial plate and rupture of chromosomes) upon mitoses in cultures of connective- tissue cells of the rabbit brought about by addition of testosterone and 3:4- benzpyrene, and to a less effect by oestrone and methylcholanthrene. Tests with other compounds of similar solubility have not yet been made. Soresina (803) describes the characters in tissue culture of a sarcoma induced in the rat by 3:4-benzpyrene and the effect of portions of this tissue when added to cultures of chick embryo cells. Willmer and Wallersteiner (844) examined the effect of a large number of aldehydes upon chick periosteal fibroblasts and cells of a dibenzanthracene chicken sarcoma growing in zlitro, and found no indication that the sensitivity to the inhibitory action of these compounds differed in the two types of cell. Morigami (694, 695) describes the growth in tissue culture of hepatoma tissue induced in a rat by p-dimethylaminoazobenzene (butter yellow) in the food. After five or six subcultures the stroma had disappeared, leaving epithelial cells only, which grew as transplantable hepatoma when implanted subcutaneously in rats. The same author (694) examined the action of this compound, and of various fractions of rice-husk oil, upon tissue cultures of chick embryo heart and 3 :4-benzpyrene sarcoma ( ? species). 5 70 J. W. COOK AND E. L. KENNAWAY

X. Characters and Composition of Tumours Stuart McDonald and Woodhouse (676) examined the growth in the thigh muscles of the mouse of transplants of sarcoma produced in this situation by injection of 1 :2 :5 :6-dibenzanthracene in lard. The primary tumours were spindle-celled with an abundant intercellular argyrophil reticulum. Trans- plantation of one of these tumours was carried on through 85 generations during thirty-seven months. Grafts were examined from one to thirty-six days after inoculation. The vascular system was studied by injection of “ hydrocollag.” Cytoplasmic glia fibrils extending beyond the cell boundary, an intercellular reticulum staining with aniline-blue, and a well-defined argyro- phi1 intercellular reticulum were demonstrated. There was abundant infiltration of muscle, but there was no invasion of bone deep to the periosteum, of perineurium or of the dermis, and the regional lymph nodes were not affected. Metastases in the lung occurred in three mice bearing “ grafts of more than thirty days development.” Cell-free filtrates of grafted tumours gave negative results on inoculation. *Duringthe first four days there is leucocytic infiltration, chiefly polymorph, around the graft. There is considerable hyperaemia, due apparently to the opening up of pre-existent channels, reaching its maximum between the eighth and twelfth days. Necrosis develops from the thirteenth day onwards. Warren Lewis (644) examined the histologic characters of 50 sarcomas produced in pure strain mice by one subcutaneous injection of 0.8 mg. 1:2 :5 :6- dibenzanthracene in lard or olive oil. “ The tumours were of usable size at from 85 to 183 days after the injection . . . all gave 100 per cent growth in mice of the strain of origin. . . .” Twenty-eight of these tumours showed principally spindle cells; the remaining 22 showed also modified skeletal muscle fibres, muscle giant cells, and myoblasts. “The muscle elements, with rare exceptions, disappear in later generations.” The nature of these muscle elements is discussed. Tumours which produce considerable neutrophil leucocytosis (294) show no distinctive characters. Lewis’ paper is illustrated by 20 photomicrographs.

TABLEXXXI: Mifofic Idex, &wth Rate, and Latent Pcrwd of Induced Tumours (Brucs, WCinm, and Andnmnrt) .-

of cells Dam required for Dam requlred for Dayr required for tumour to double tumour to increaac turnour r0 increw latent pedod In mitds in size from 10- to 0.1 cma from 10- to 0.1 cm* (days)

0.700.96 I 4.1-13 .O 10980 60

Brues, Weiner, and Andervont (446) investigated the correlation between the malignancy of chemically induced tumours, as measured by growth rate and mitotic index, and the length of the latent period before the appearance of palpable tumours. Mice of various inbred strains received subcutaneous injections of carcinogenic agents, “ MCA ” and ‘‘ DBA *’(presumably methylcholanthrene and 1 :2 :5 :6-dibenzanthracene, but this is not stated) * in cholesterol or lard, and record was kept of the time of first appearance of a palpable CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 571 tumour, the rate of increase of measurements, and mitosis-per 1000 or more tumour cells when the mice were killed. The correlation found between these data is expressed in Table XXXI. Dobrovolskaya-Zavadskaya and Nekhorocheff (504) studied the rate of growth of spontaneous cancers of the mouse and of ten sarcomas induced by 1 :2 :5 :6-dibenzanthracene. A linear rate of growth was found in 88 per cent of the cases. Pollia (746) records a loss in vigour of growth in successive grafts of a fibrosarcoma induced in a rat by 1: 2 :5 :6-dibenzanthracene, while the grafts of a spontaneous uterine tumour arising in the same strain of rats showed increased vitality. Kishi, Fujiwara and Nakahara (600) compared the chemical composition of the normal liver of the rat with that of grafted hepatoma induced originally in the organ by 4’-amino-2 :3’-azotoluene. They say: With hepatoma cholesterol at least another sterol constantly crystallizes together when recrystallized from alcohol, and a comparison sf..%.... absorption spectra demonstrated that about 1/15 of hepatoma cholesterol consl$ed.d dtjpsteDQ\gr 7-dehydrocholesterol, i.e. provitamin D. Wit4 choies’tidl from normal rat liver the coexistence of ergosterol or 7-dehy&&61~$6tcrt.$as not f&d Hpect3.ogmphically.” The iodine value of the p&~pk&&?d hfatonla b&% About %If that found in normal liver, hence the fatty acidgot )&$@ho\its.are more saturated. Hepa- toma contained about one-third (;f*fie’ih%’n%*~ftotal fatty acids found in normal liver. ‘‘ The quantitative difference was entirely due to that of phosphatide between the two tissues.”

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589. IBALL, J.: Ztschr. Kristal. (A) 94: 7, 1936. 590. IBALL,J.: Ztschr. Kristal. (A) 99: 230, 1938. 591. IBALL,J.: Ztschr. Kristal. (A) 100: 234, 1938. 592. IBALL,J.: Am. J. Cancer 35: 188, 1939. 593. IKI, H.: Gann. 33: 216, 1939. 593A. ITO,S.: Gann. 33: 453, 1939. 594. JEDLICKY,A., LIPSCHUTZ,A., AND VARGAS,L.: Compt. rend. SOC.de biol. 130: 1466, 1939. 595. JONES,W. E., AND UMACE,G. R.: J. Chem. SOC.,1938, p. 1853. 596. JOSEPH,L.: Proc. SOC.Exper. Biol. & Med. 41: 334, 1939. 597. JUDINA,N. D.: J. mbd. Ukraine 7: 819, 1937. (Read in abstract only. Chem. Abstr. 32: 7993, 1938.) 598. JUDINA,N. D.: J. mbd. Ukraine 8: (a) 149; (6) 625, 1938. (Read in abstract only. Chem. Abstr. 33: 8278, 1939.) 599. KISEI, S., FUJIWARA,T., AND NAKAHARA,W.: Gann 32: 469, 1938. 600. KISBI, S., FUJIWAJU, T., AND NAKAHARA,W.: Gann 33: 332, 1939. 601. Km, E.: Klin. Wchnschr. 17: 1279, 1938. 602. KLEINENBERC,G. E.: Arch. sci. biol. U.S.S.R. 51: 127, 1938. (Read in abstract only. Chem. Abstr. 33: 6429, 1939.) 603. KLING,A., AND HEROS,M.: Compt. rend. Acad. d. sc. 208: 2075, 1939. 604. KLINC,A,, SAMSSONOW,N., AND HEROS,M.: Compt. rend. Acad. d. sc. 206: 1268, 1938. 605. KLINC,A., SAYSSONOW,N., AND HEROS,M.: Bull. Acad. de mbd., Pans, 119: 439, 1938. 606. KLINKE,J.: Ztschr. f. Krebsforsch. 46: 334, 1937. 607. KLINKE,J.: Ztschr. f. Krebsforsch. 47: 341, 1938. 608. KLINKE,J.: Ztschr. f. Krebsforsch. 47: 348, 1938. 609. KOCH, c., SCHREIBER, B., AND SCHREIBER, G.: Bull. hot. franc. p. l’htude du Cancer 28: 852, 1939. 610. Row, O., LIPSCHUTZ,A,, AND VARGAS,L.: Compt. rend. SOC.de biol. 130: 303, 1939. 611. KORENCHEVSKY,V., AND HALL,K.: J. Obst. & Gynaec. Brit. Emp. 45: 22, 1938. 612. KOBENCHEVSXY,V., AND HALL,K.: Nature 142: 998, 1938. 613. KORENCHEVSKY,V., AND HALL,K.: Nature 144: 1048, 1939. 614. KORTEWEC,R., AND THOMAS,F.: Leeuwen.-Ver. 4: 44, 1937. 615. KORTEWEC,R., AND THOMAS,F.: Am. J. Cancer 37: 36, 1939. 616. KRISHNAN,R. S., AND BANERJEE,S.: Phil. Trans. Roy. SOC.,London, Ser. A, 234: 265, 1934-35. 617. KRISHNAN,K. S., AND BANERJEE,S.: Ztschr. f. Kristal. (A) 91: 170, 1935. 618. KRISHNAN,K. S., AND BANERJEE,S.: Ztschr. f. Kristal. (A) 91: 173, 1935. 619. LACASSACNE,A.: Bull. Assoc. franq. p. I’btude du cancer 27: 96, 1938. 620. LACASSACNE,A.: Bull. Assoc. franc. p. l’btude du cancer 28: 951, 1939. 621. LACASSACNE,A.: Canad. M. A. J. 37: 112, 1937. 621A. LACASSACNE,A.: Ergebnisse der Vitamin- und Hormonforsch. 2: 259, 1939. 622. LACASSACNE,A.: Am. J. Cancer 37: 414, 1939. 623. LACASSACNE,A., AND DANYSZ,S.: Compt. rend. SOC.de biol. 132: 395, 1939. 624. LACASSACNE,A.: Compt. rend. SOC. de biol. 129: 641, 1938. 625. LACASSACNE,A.: Compt. rend. SOC.de biol. 130: 591, 1939. 626. LACASSACNE,A.: Compt. rend. SOC.de biol. 132: 222, 1939. 627. LACASSACNE,A.: Compt. rend. SOC.de biol. 132: 365, 1939. 628. LACASSACNE,A., AND RAYNAUD,A.: Compt. rend. Soc. de biol. 131: 586, 1939. 629. LACASSACNE,A., AND RAYNAUD,A.: Compt. rend. SOC.de biol. 132: 431, 1939. 630. LAMBERT,P., AND LECOYTE,J.: Compt. rend. Acad. sc. 208: 1148, 1939. 631. LANZA,G.: Pathologica 30: 185, 1938. 632. LARIONOW,L. T., IVACHENTZOVA,N. G., AND TCHERTKOVA,M. A.: Bull. de biol. et de mbd. er.6: 113, 1938. 633. LARIONOW,L. T., AND SOBOLEVA,N. G.: Vestnik rentgen. i radiol., Leningrad, 20: 276, 1938. 634. LA=, H. J., HIIDEBBAND,E., AND SCHOCKE,H.: Ztschr. f. d. ges. exper. med. 105: 370, 1939. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 577

635. LAUFFER,M. A., AND STANLEY,W. M.: J. Biol. Chern. 123: 507, 1938. 636. LAWRENCE,J. H.,AND GARDNER,W. U.: Am. J. Cancer 33: 112, 1938. 637. LEROY,G. V., KANDEL,E. V., AND BRUNSCHWIG,A,: Am. J. Cancer 32 : 449, 1938. 638. LEVINE,M.: Proc. SOC.Exper. Biol. & Med. 40: 599, 1939. 639. LEWIS,M. R.: Am. J. Cancer 34: 399, 1938. 640. LEWIS,M. R.: Am. J. Cancer 34: 431, 1938. 641. LEWIS,M. R.: Am. J. Cancer 36: 34, 1939. 642. LEWIS,M. R.,AND LICHTENSTEIN,E. G.: Growth 1 : 375, 1937. 643. LEWIS,W. H.: Am. J, Cancer 35: 408, 1939. 644. LEWIS,W. H.: Am. J. Cancer 37: 521, 1939. 645. LEWIS,W. H.: Arch. f. exper. Zellforsch. 23: 1, 1939. 646. LEWIS,W. H.: Arch. f. exper. Zellforsch. 23: 8, 1939. 647. LIBERTI,V.: Riv. di pat. sper. 22: 307, 1939. (Read in abstract only. Chem. Zentralbl. 1: 1066, 1940.) 648. LIPSCHVTZ,A.: Gynkc. et obst. 36: 407, 1937; 37: 17, 1938. 649. LIPSCHUTZ,A,, AND IGLESIAS,R.: Compt. rend. SOC.de biol. 129: 519, 1938. 650. LIPSCHUTZ,A., IGLESIAS,R., AND VARGAS,L.: Compt. rend. SOC, de biol. 130: 1536, 1939. 651. LIPSCHUTZ,A., RODRIGUEZ,F., AND VARGAS,L.: Compt. rend. SOC.de biol. 130: 939, 1939. 652. LIPSCHUTZ,A., AND VARGAS,L.: Compt. rend. SOC.de biol. 130: 9, 1939. 653. LIPSCHUTZ,A., AND VARGAS,L.: Compt. rend. SOC.de biol. 130: 596, 1939. 654. LIPSCHUTZ,A,, AND VARGAS,L.: Compt. rend. SOC.de biol. 131: 27, 1939. 655. LIPSCHUTZ,A., AND VARCAS, L.: Lancet 1: 1313, 1939. 656. LIPSCHBTZ,A., VARGAS,L., AND IGLESIAS,R.: Compt. rend. SOC. de biol. 129: 524, 1938. 657. LOEB,L., SUNTZEFF,V., AND BURNS,E. L.: Am. J. Cancer 34: 413, 1938. 658. LONSDALE,K.: Proc. Roy. SOC.,London, Ser. A. 159: 149, 1937. 659. MACATH,M. A.: Acta Med. URSS 2: 53, 1939. 660. MACNUS,H. A.: J. Path. & Bact. 49: 21, 1939. 661. MAISIN,J.: Acta, Unio internat. contra cancrum 4: 704, 1939. 662. MAISIN, J., POURBAIX,Y., AND CAEYMAEX,P.: Compt. rend. SOC.de biol. 127: 1477, 1938. 663. MAISIN,J., POURBAIX,Y., AND CAEYMAEX,P.: Compt. rend. SOC. de biol. 127: 1479, 1938. 664. MAISIN,J., POURBAIX,Y., AND CAMERAN,J.: Compt. rend. SOC.de biol. 128: 806, 1938. 665. MAISIN,J., POURBAIX,Y., AND CAMERAN,J.: Compt. rend. Soc. de biol. 130: 1381, 1939. 666. MAXSIN,J., POURBAIX,Y., AND CAMERAN,J.: Compt. rend. SOC.de biol. 132: 87, 1939. 667. MAISIN,J., POURBAIX,Y., AND CUVELIER:Compt. rend. SOC.de biol. 132: 315, 1939. 668. MAISIN,J., POURBAIX,Y., AND RIJCKAERT,G.: Compt. rend. SOC. de biol. 130: 109, 1939. 669. MAISIN,J., POURBAIX,Y., AND VANDE VOORDE,C.: Compt. rend. SOC.de biol. 132: 86, 1939. 670. MAISIN, J., AND Yu HANTANG: Compt. rend. SOC.de biol. 132: 313, 1939. 671. MASAYAMA,T., IKI,H., YOKOYAMA,T., AND HASIMOTO,M.: Gann 32 : 303, 1938. 672. MASAYAMA,T., AND YOKOYAMA,T.: Gann 33: 214, 1939. 673. MAUER,G.: Arch. f. exper. Zellforsch. 21: 191, 1938. (Read in abstract only. Chem. Abstr. 32: 8540, 1938.) 674. MAY,H., AND LITZKA,G.: Ztschr. f. Krebsforsch. 48: 376, 1939. 675. MCCLUSKE,J. A. W., AND NIVEN,J. S. F.: J. Path. & Bact. 47: 155, 1938. 676. MCDONALD,S., JR., AND WOODHOUSE,D. L.: J. Path. & Bact. 47: 615, 1938. 677. MCEUEN,C. S.: Am. J. Cancer 34: 184, 1938. 678. MCEUEN,C. S.: Am. J. Cancer 36: 551, 1939. 679. MCINTOSH,J.. AND SELBIE,F. R.: Brit. J. Exper. Path. 20: 49, 1939. 680. MCJUNKIN,F. A,, AND WOLAVKA,W.: Arch. Path. 25: 506, 1938. 681. MELLANBY,E.: J. Path. & Bact. 46: 447, 1938. 5 78 J. W. COOK AND E. L. KENNAWAY

682. MEYER,K. H.: Annalen der Chemie 379: 73, 1911. 683. MIDER,G. B., AND MORTON,J. J.: Am. J. Path. 15: 299, 1939. 684. MIDER,G. B., AND MORTON,J. J.: Am. J. Cancer 37: 355, 1939. 685. MIDER,G. B., AND MORTON,J. J.: Proc. SOC.Exper. Biol. & Med. 42 : 583, 1939. 686. MIKHAILOV,B. M., AND TSCRERNOVA,N. G.: Compt. rend, Acad. sc. URSS 20: 579, 1938. (Read in abstract only. Chem. Abstr. 33: 5842, 1939.) 687. MIZUTA,T.: Gann 33: 221, 1939. 688. VON MOELLENDORF,W.: Klin. Wchnschr. 18: 1098, 1939. 689. MOXLER,H., AND SORGE,J.: Helv. Chim. Acta. 22: 229, 1939. 690. MORELLI,E., AND DANSI,A.: Nature 143: 1021, 1939. 691. MORELLI,E., AND GUASTALLA,A.: Boll. lega ital. per la lotta contro il cancro 10: 110, 1936. 692. MORELLI,E., AND GUASTALLA,A.: Chim. e Ind. 37: 1141. 693. MORICARD,R., AND CAUCHOIX,J.: Compt. rend. SOC. de biol. 129: 556, 1938. 694. MORICAMI,S.: Gann 32: 389, 1938; 33: 250, 281, 384, 1939. 695. MORIGAMI,S.: Jap. J. M. Sc., Part V (Pathology) 4: 107, 1939. 696. MORTON,J. J., AND MIDER,G. B.: Science 87: 327, 1938. 697. MORTON,J. J., AND MIDER,G. B.: Proc. SOC.Exper..Biol. & Med. 41: 357, 1939. 698. MOSETTIG,E., AND KRUEGER,J. W.: J. Org. Chem. 3: 317, 1938. 699. MOTTRAM,J. C.: Am. J. Cancer 32: 76, 1938. 700. MOTTRAM,J. C.:Nature 144: 154, 1939. 701. MOTTRAM,J. C.: Proc. Roy. SOC.,London, ser. B, 126: 560, 1939. 702. MOTTRAM,J. C., AND DONIACH,I.: Lancet 1: 1156, 1938. 703. NAGAO,N.: Proc. Imp. Acad. Tokio 15: 321, 1939. Gann 34: 13, 1940. 704. NAYAIIARA,W., AND FUJIWARA,T.: Gann 32: 477, 1938. 705. NAKAHARA,W., FUJIWARA,T., AND MORI,K.: Gann 33: 57, 1939. 706. NA~ABARA,W., Mom,K., AND FUJJWARA,T.: Gann 32: 465, 1938. 707. NAIAHARA,W., MORI,K., AND FUJIWARA,T.: Gann 33: 13, 1939. 708. NAYAIIARA,W., Mom, K.,AND FUJJWARA,T.: Gann 33: 406, 1939. 709. NAWMURA,H., YAMADA,M., OHAMURA,H., YODA,Y., AND NIBS, T.: Gann 32: 245. 1938. 710. NAOATANI,M., NAKANO,K., AND Ow,Y.: Gann 32: 2-40, 1938. 711. NATHANSON,I. T., AND ANDERVONT,H. B.: Proc. Soe. Exper. Biol. 8 Med. 40: 421, 1939. 712. NELSON,W. 0.: Endocrinology 24: 50, 1939. 713. NEWYAN,M. S.: J. Am. Chem. SOC.60: 1141, 1938. 714. NEWYAN,M. S.: J. Am. Chem. SOC.60: 1368, 1938. 715. NEWYAN,M. S.: J. Am. Chem. SOC.60: 2947, 1938. 716. NEWYAN,M. S., AND JOBHEL, L. M.: J. Am. Chem. SOC.60: 485, 1938. 717. NEWMAN,M. S., AND ORCHIN,M.: J. Am. Chem. SOC.60: 586, 1938. 718. NEWMAN,M. S., AND ORCHIN,M.: J. Am. Chem. Soc. 61: 244, 1939. 719. NICOD,J. L., AND REGAMEY,J.: Bull. Assoc. franc. p. l'itude du cancer 27: 706, 1938. 720. NISHNAMA,Y.: Gann 32: 85, 1938. 721. NOBLE,R. L.: Lancet. 2: 192, 1938. 722. NOBLE,R. L.: J. Endocrinology 1: 216, 1939. 723. OBERLING,C., Cuhxu, M., AND GU~RIN,P.: Bull. Amc. franc. p. 1'Ctude du cancer 28: 198, 1939. 724. OBERLXNC,C., G~RIN,P., AND Guhnr, M.: Compt. rend. SOC.de biol. 130: 417, 1939. 725. OBERLING,C., GU~IN, P., G~IUN,M., AND SANN~,C.: Compt. rend. SOC.de biol. 130: 17, 1939. 726. OBEILLING,C., SANN~,C., GU~RIN,M., AND GuhuN, P.: Leeuwen.-Ver. 4: 57, 1937. 727. OBERLING, C., SANNIJS, C., G~RIN,P., AND GUERIN,M.: Compt. rend. SOC. de biol. 131: 455, 1939. 728. Om,J. W.: J. Path, & Bact. 46: 495, 1938. 729. Onn, J. W.:J. Path. & Bact. 49: 157, 1939. 729A. Onn, J. W., AND STICKLAND,L. H.: Chemistry & Industry 58: 1088, 1939. 730. Om,J. W., AND POLSON,C. J.: Am. J. Cancer 32: 114, 1938. CHEMICAL COMPOUNDS AS CARCINOGENIC AGENTS 5 79

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Papers Read in Abstract Only After the Conrpletion of the Bibliography

BENECKE,E., AND SCHRODER,J. : Ueber experimentelle Geschwulsterzeugung in Speichel- driisen, Ztschr. f. Krebsforsch. 49: 505, 1939. HOPPER,S. H.,AND CLAPP,D. B.: Effect of carcinogenic and other hydrocarbons on the growth of Escherichia communior, J. Bacteriol. 38: 13, 1939. LEBENZON,E. G. : Atypical mitoses caused by carcinogenic hydrocarbons in tissue cultures, J. med., Ukraine 8: 713, 1938; Chem. Abstr. 33: 8278, 1939. NEUMAN,I. M.: Alterations of tissue metabolism under the influence of 1 :I-benzpyrene, Arch. sc. biol. URSS 51: 69, 1938. SHABAD,L. M., AND URINSON,J, P.: Alterations in the liver of guinea-pigs, following administration of a chemically pure cancerogenic substance (1 :2 :5 :6-dibenzanthracene), Arch. sc. biol. URSS 51: 105, 1938. TUNODA,R.: Studien iiber die Beziehungen zwischen den Hormonen und malignen Tumoren. I. Mitt. Ueber den Einfluss von Follikelhormon und Kastration auf die Entstehung des durch Buttergelb erzeugten Rattenhepatorns, Mitt. jap. Ges. Gynak. 34: 46, 1939.

APPENDIXTO BIBLIOGRAPHY Earlier Literature of Carcinogenic Compounds BLOCH,B. : Zur Chemie und Biologie des experimentellen Teerkrebses, Leeuwen-Ver. 1 : 46, 1922. BURROWS,H., AND KENNAWAY,N. M.: On some effects produced by applying oestrin to the skin of mice, Am. J. Cancer 20: 48, 1934. CLAR,E.: Zur Kenntnis mehrkerniger aromatischer Kohlenwasserstoffe und ihrer Abkomm- linge, I: Dibenzanthracene und ihr Chinone, Berichte d. deutsch. chem. Gesellsch. 62 : 350, 1929. 582 J. W. COOK AND E. L. HENNAWAY

COOK,J. W.: Polycyclic aromatic hydrocarbons, Parts I-V, J. Chem. SOC., 1930, p. 1087; 1931, pp. 487, 489, 499, 2012. COOK,J. W.,HEWETT, C., AND HIIE~E~,I.: Coal tar constituents and cancer, Nature 130: 926, 1932. FIESEB,L. F., AND DIETZ,E. M.: Beitrag zur Kenntnis der Synthese von mehrkernigen An- tracenen, Berichte d. deutsch. chem. Geselbch. 62: 1827, 1929. HXECEB,I.: The spectra of cancer-producing tars and oila and of related substances, Biochem. J. 24: 505, 1930. KENNAWAY,E. L. : Further experiments on cancer-producing substances, Biochem. J. 24 : 497, 1930. KENNAWAY,E. L., AND HIEGEB,I.: Carcinogenic substances and their fluorescence spectra, Brit. M. J. 1: 1044, 1930. LACASSACNE,A.: Mdtaplade dpidermoide de la prostate provoqude chec la sou&, par des injections r4p6tCes de fortes doses de folliculine, Compt. rend. SOC.de biol. 113: 590, 1933. (1 WEITZENB~IC,R., AND KLLNGLER,A.: Synthese der isomeren Kohlenwawrstoffe 1 :2:5:6- Dibenzanthracen und 3:4: S:BDibenzphenanthren, Monatshefte f. Chemie 39: 315, 1918.