Pulmonary Tumors in Experimental Animals

Pulmonary Tumors in Experimental Animals MICHAEL B. SHIMKIN National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Page I. Historical Introduction. .................... . . . . . . . . . . . 223 11. Frequency and Distribution of Pulmonary T u . . . . . . . . . . . . 225 111. Pulmonary Tumors in Other Animals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 IV. Morphology and Biochemistry of Pulmonary Tumors in .Mice. . . . . . . . . . . 229 1. Gross and Microscopic Appearance.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 2. Biochemical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. IIistogenesis of Pulmonary Tumors in LVice... . . . . . . . . . . . . . . . . . . . . . . . . 233 VI. Influence of Heredity in Pulmonary Tumors in Micc.. . . . . . . . . . . . . . . . . . 235 VII. Polycyclic Hydrocarbons and Related Compounds. . . . . . . . . . . . . . . . . . . . . 237 VIII. Urethane and Related Compounds.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 IX. Other Chemical and Physical Agents, Including Inhalants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 1. Miscellaneous Agents.. . 2. Exposure to Inhalants.. . . . . . . . . . . . . . . . . . . . . . . . . . . S. Factors Affecting Pulmonary Tumor Induction in Mice.. . . . . . . . . . . . . . . 248 1. Sex, Age, Diet, and Inflammation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 2. Pulmonary Tumors in Embryo Mice.. . . . . . . . . . . . . . . . . . . XI. Mechanism of Induction of Pulmonary Tumors in Mice.. . . . . 1. Mode of Action of the Carcinogen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 2. Mode of Reaction of the Lung. . . . . . . . . . . . . . 254 XII. Pulmonary Tumors in Man and General Discussion.. . . . . . . . . . . . . . . . . . . 256 1. Bronchogenic Carcinoma, . . . . . . . . . . . . . . . . . . . . . . . . . . 256 2. Other Pulmonary Tumors.. . . . . . . . . . . . . . . . . . . . . . . . . 258 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

I. HISTORICAL INTRODUCTION This review summarizes investigations concerning primary pulmonary neoplasms in animals, and selectively covers the literature from 1896 to 1953. The terms of reference exclude metastatic neoplasms of the lung, and more generalized neoplasms with incidental localization in the lung, such as lymphosarcoma or hemangioendothelioma. Most of the experimental work has been performed with the adenomatous pulmonary tumor of the mouse, and most observations and conclusions perforce must be limited to mice. Recent reports on the induction of pulmonary tumors in rats and in guinea pigs allow the extension of the investigations to other species. Reports on pulmonary tumors in larger animals are limited to descriptions of individual cases and a few, mostly unsuccessful, attempts t o induce the tumors. 223

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Cancer research, as all biological research, is dependent upon the availability of proper materials and techniques for its work. The chief material has been the mouse, and the chief techniques have been the transplantation and the induction of neoplasms. On this basis, a large portion of the history of cancer research can be divided into three main periods. The period 1890 to 1918 can be designated as the morphologytransplantation era. The period of 1918 to 1935 can be designated as the genetics-tar tumor era. The modern period, since 1935, dates from the general use of homozygous mice and the galaxy of defined chemical agents for the induction of experimental neoplasms. Studies on pulmonary tumors in mice reflect these three periods of research on cancer. The first report of a primary pulmonary tumor in a mouse is usually attributed to Livingood (116) in 1896. During the next fifteen years, a large number of cases were collected and described by Murray (1 43), Tyzzer (1 96) , Jobling (93), and Haaland (63). Slye, Holmes, and Wells (183) in 1914 published the findings on the first six thousand autopsies of mice in their colony. Among these were 160 mice with pulmonary tumors, of which 63 were considered histologically malignant and 4 had metastasized. During the tar-painting era of cancer research, Murphy and Sturm (144)in 1925 first clearly demonstrated the induction of primary pulmonary tumors with tar, at the same time avoiding the appearance of skin carcinomas. With the availability of inbred strains of mice, Lynch (124) in 1926 showed th at different strains had markedly different frequencies of pulmonary tumors and that susceptibility to tar-induced pulmonary tumors paralleled the spontaneous frequency. She applied the material to genetic studies on induced and spontaneous neoplasms. With the advent of carcinogenic polycyclic hydrocarbons, it was soon reported that the injection of these compounds into mice also markedly increased the frequency of pulmonary tumors. Andervont (1-18) in 1935 initiated extensive studies, and his important contributions dealt with a comparison of strain susceptibility, the effects of heredity, various carcinogens and modes of administration, and the successful serial transplantation of the tumors. Shimkin (171) in 1940 introduced more exact quantitative methods by considering the number of nodules in the lungs. Grady and Stewart (56) in 1940 rlarified the problem of the histogenesis of the neoplasm. Heston (69-72) in 1940 began his gene-linkage studies on the tumor, which remain among the better contributions to experimental work on the inheritance of cancer. Nettleship, Henshaw, and Meyer (145) in 1943 stimulated wide interest in their discovery that urethane induced pulmonary tumors in mice. The work was extended along chemical lines by Larsen (102-104) and to the induction of pulmonary tumors

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in rats by Jaff6 (91, 92). The recent work of W. E. Smith (185, 187) on pulmonary tumors induced in embryonic lungs of mice is certain to be of continuing importance. In the investigations of pulmonary tumors in mice, three groups in three institutions have made particularly noteworthy and continually productive advances. The Imperial Cancer Research Fund in England laid the morphological foundations. The Rockefeller Institute for Medical Research in New York initiated the studies on induction and inheritance of the tumors. The National Cancer Institute in Bethesda has been most strongly represented in the advances since 1937.

11. FREQUENCY AND DISTRIBUTION OF PULMONARY TUMORS IN MICE The original investigators using mice noted that mammary tumors and primary pulmonary tumors were the most frequent types of neoplasms in the species. Nevertheless, the frequency of pulmonary tumors is relatively low in unselected, nonhomozygous mouse populations. Twort and Twort (195), in a study of 60,000 mice, stated that the frequency was approximately 1%. Wells, Slye, and Holmes (20l), in 147,132 autopsies of mice, found pulmonary tumors in 2865, or 2%. Andervont (14) studied 34 wild house mice and their progeny and found 6 with pulmonary tumors, a t the age of 20 to 23 months. Tumors of the lung are seldom found in noninbred mice under one year of age and occur in the same frequency among males and females. The presence of pulmonary tumors is not associated with parasitic infestations, nor is there any evidence of communicability or infectious transmission between animals. With the development and general use of inbred mice, it was found that various strains had markedly different frequencies of pulmonary tumors. Since inbreeding and selection of strains toward homogeneity were made without particular reference to the occurrence of pulmonary tumors, the segregation of this characteristic in specific strains was unpremeditated. In retrospect, therefore, there must have been connection between certain definite genic characters and the susceptibility to pulmonary tumors. Since 1935, data have been accumulating on the frequency and distribution of pulmonary tumors in the various homozygous strains of mice that are used in cancer research. It is notable that the frequency of pulmonary tumors in a strain appears to be more constant, as reported by various laboratories, than the frequency of mammary or hepatic tumors. This would indicate that pulmonary tumors are less under the influence of various environmental factors than many other neoplasms

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of the mouse. It is now also clear th at the frequency of pulmonary tumors involves the factors of time of appearance and multiplicity; i.e., in more susceptible strains, the tumors appear a t an earlier age and a higher proportion of animals have multiple tumors, whereas in the resistant strains, only rare single tumors appear in animals that are 18 months of age or older. The classical most susceptible strain of mice relative to pulmonary tumors is strain A, established by Strong (192) in 1921. Pulmonary tumors that are recognizable grossly are seen in mice as young as 3

2 4

6

8 10 12 14 16 18 20 22 24 Age in months

FIG.1. Frequency of pulnionary tumors in three inbred strains of mice, a t different ages. Data for strain A mice from Shimkin ( l i l ) , Heston (71), and Bittner (24); for strain C (Bagg alb C) from -4ndervont (13); for strain CJEIfrom Andervont (12).

months, and the frequency rises steadily to approximately 90% by 18 months (Fig. 1). Two or more tumors, but usually not more than five, begin t o appear in animals 8 months of age. The Swiss albino mice are apparently as susceptible to pulmonary tumors as strain A (57, 152). Bagg albino C, I, Y, and C3H are of intermediate susceptibility in the approximate order given, showiiig pulmonary tumors in 10% to 30% of the animals over a year old (7, 12, 13); tumors are not observed in mice under 6 months of age and multiple tumors are infrequent. Strains C57 black and (‘57 leaden (L or 31) are most resistant, having practically no pulmoriary tumors even when the mice are two years of age. Little ct uZ. (115) recorded one pulmonary tumor in 742 mice of the C57 black strain. Table I presents data on the frequencies of pulmonary and of mammary tumors in nine commonly used strains of mice. There is no relationship between the frequencies of these two types of iteoplasm, and there

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appears to be no relationship of frequency or of susceptibility to induction of pulmonary tumors to any other spontaneous or induced neoplastic process, such as hepatoma, lymphoma, subcutaneous sarcoma, or cutaneous carcinoma. The essential identity of spontaneous and of induced pulmonary t.umors is further attested by the fact that the frequency, time of appearance, and multiplicity of induced tumors are in parallel with the spontaneous occurrence of the neoplasms. To all induction procedures, strain TABLE I Frequency of Pulmonary and Mammary Tumors in Eight Strains of Mice&

Strain

A Swiss B alb C (C) I Y C,H dba C57leaden (L or M) Cg7black 0

Pulmonary Tumors Per Cent of Animals 12-18 Months Old

Mammary Tumors Per Cent of Breeding Females

70-90 40-50 15-25 10-20 10-20 5-1 5 5 <1
70-85 20 5 1 5 75-100 55-75 <1 <1

Modified from Heston (73).

A mice have proved to be most susceptible, and strains CWblack and C6, leaden the least susceptible, with the intermediate strains retaining their relative position. This observation has greatly facilitated many investigations in that observations can be made within a few weeks with induced tumors instead of within the 18 to 24 months required for studies on spontaneous tumors. It is important to note that Shapiro and Kirschbaum (167), in a study of seven strains of mice, recorded that the NH strain, stated to have a very low spontaneous frequency of pulmonary tumors, was susceptible to the induction of such tumors with urethane. Since the actual frequency of spontaneous pulmonary tumors in the NH strain at various ages has not been published, this seeming exception should be considered sub judice at the present time. 111. PULMONARY TUMORS IN OTHER ANIMALS It is a commonly accepted impression that primary pulmonary tumors are rare except in the mouse and in the human being. The conclusion regarding mice is accentuated by the high frequency observed in some inbred strains. In unselected populations it is approximately 2 % when

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the animals are maintained for their whole life span. In man, the frequency among those dying of all causes does not exceed 2 % (198)even considering the marked rise in frequency among males during the past 30 years. Pulmonary tumors in laboratory animals other than the mouse are stated to he rare. McCoy (13-1)found no pulmonary tumors among autopsies on 100,000 wild rats, a population in which rats of advanced age undoubtedly were exceptional. Saxton et al. (156)observed two pulmonary adenomas among 498 rats of the Osborne-Mendel strain-a frequency of 0.4%. Horn and Stewart (87) reported a pulmonary tumor, histologically identical with the mouse neoplasm, in a one-year-old rat of Marshall strain 520 as the only example of a spontaneous pulmonary tumor in a rat at the Xational Cancer Institute. In regard to pulmonary tumors in rats, it should be noted that lungs of old rats often have areas of inflammation and bronchial metaplasia that can be readily confused with neoplastic reactions (149). Only individual cases of pulmonary tumors have been recorded in guinea pigs, and this species is supposed to have very few spontaneous neoplasms of any type. Goldberg (55)described a single adenocarcinoma of the lung in a guinea pig, and Norris (146) published an occurrence of pulmonary adenomatosis in one animal. One pulmonary tumor was found by Heston and Deringer (81)in a guinea pig of inbred strain 2;the tumor resembled the pulmonary tumor of the mouse. Lorenz et aZ. (120a) recently reported three cases of pulmonary tumors in 19 guinea pigs three to six years of age. Polson (150)recorded one carcinoma of the lung among 66 instances of neoplasms in rabbits. Sjolte (181)included one adenocarcinoma of the lung in one rabbit. Schinz (162,163) reported two instances of adenocarcinoma of the lung in rabbits. One had received 0.1 g. of cobalt into the thigh four years previously, and the other was a female that five years before had been injected with methylcholanthrene into the pregnant uterus. Whether these are instances of induced or of spontaneous neoplasms is anyone’s guess. The only example of a pulmonary tumor in a fowl was the case of Apperly (19),in a Black Orpington over one year of age, that had an adenocarcinoma of the lung with metastases to the liver. Although it is impossible not to conclude that rats, guinea pigs, rabbits, and fowl develop pulmonary tumors much less frequently than mice, one cannot but wonder to what extent the age of the animals at the termination of most experiments, the preponderant interest of the investigators in other neoplasms, and lack of care in the autopsy examination contribute to this rarity.

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Sticker (191) in 1902 tabulated extensive data regarding carcinomas of larger domestic animals, primarily from veterinary and abattoir sources in Germany from 1858 to 1900. He included 13 of the lung among 311 carcinomas of the horse, 3 of the lung among 73 carcinomas in bovines, 10 of the lung among 766 carcinomas in dogs, 3 of the lung among 121 carcinomas of the cat, and 1 carcinoma of the lung among 7 carcinomas in sheep. Feldman (52) in his monograph which gives many primary sources added 3 pulmonary carcinomas among 41 carcinomas in sheep, and 1 in a kangaroo. Sjolte (181) reviewed the subject of primary pulmonary cancer in animals and presented 23 cases from Copenhagen, of which 10 were in the dog, 4 in the horse, 7 in cattle, and 1 in a jaguar. Ten of the neoplasms were adenocarcinomas, 6 were solid carcinomas, 3 were epidermoid carcinomas, and 3 contained sarcomatous elements and were designated as adenocarcinosarcoma. Metastases of the tumors were stated to follow the same distribution as was to be anticipated in man. It is difficult, if not impossible, to indicate the true frequency of pulmonary neoplasms in various species of animals, especially when information is lacking regarding the age distribution of the population coming to autopsy. It can certainly be concluded that pulmonary neoplasms are to be found in a wide variety of domesticated and wild animals but that the frequency is probably considerably lower than among mice and human beings. For example, Feldman (52) estimated that 8%to 10% of the older dogs are affected with neoplasms, of which carcinoma represents 40% to 50%. If we take 10 pulmonary carcinomas among 766 canine carcinomas, or 1.3% of carcinomas from Sticker’s (191) data as representative, this would indicate the frequency of the tumor among older dogs at approximately 0.05%, or less than one-twentieth of that found in the murine and the human species.

IV. MORPHOLOGY AND BIOCHEMISTRY OF PULMONARY TUMORS IN MICE 1. Gross and Microscopic Appearance

Primary adenomatous pulmonary tumors in mice have an extremely uniform gross and microscopic appearance. I n the gross, or after fixation, the tumors are pearly white, glistening, discrete round nodules, often situated just below the visceral pleura (Fig. 2). There is no predilection €or side or lobe. The tumors are sharply contrasted against the normal tissue of the lung and have a rubbery consistence. With practice, the tumors can be correctly identified with the naked eye or under a dissecting microscope when they are a fraction of a millimeter in diameter. The

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FIG.2. .\Iultiple induced tumors of the lung of mouse

FIG.3. Primary pulmonary tumor of the mouse

x

x

3.8. From Stewart (189).

60. From Stewart (189).

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presence of a superimposed pneumonic consolidation may obscure the appearance of small tumors, and the presence of a n obvious tumor elsewhere may make distinction difficult between primary pulmonary tumor and metastasis. Under the microscope, the tumor is devoid of a capsule and infiltrates and compresses the surrounding pulmonary tissue, with the intact somewhat thickened pleura over the mass (Fig. 3). The tumor is usually of

FIG.4. Primary pulmonary tumor of the mouse X 200. From Stewart (189).

a uniform adenomatous pattern, consisting of closely packed columns of cuboidal or columnar cells (Fig. 4). Papillary formation is frequent in larger tumors. The cells are uniform in size and shape, with a homogenous, acidophilic cytoplasm and round, hyperchromatic nucleus of moderate size. Cilia are not encountered. Mitotic figures are rare. The sparse stroma is composed of adult-appearing fibroblasts, and there are few blood vessels. The margins of the tumor are usually completely devoid of inflammatory reaction, lymphocytic infiltration, or increase in fibrous elements. Occasional small areas of necrosis and small cysts may be encountered in the larger masses. Well over 95% of all pulmonary tumors in mice present this appear-

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ance. There seems to be no morphological difference between the neoplasms seen in iioniribred mice and in mice of various homozygous strains (151, 170). The tumors are the same whether they are spontaneous or induced by carcinogenic agents, except, that the latter are multiple, whereas most spontaneous tumors are single. In large series of pulmonary tumors in mice, a few unusual forms have been encountered. Tyzzer (196) described one epidermoid carcinoma, and in this animal unidentified crystals were observed in the pulmonary parenchyma and bronchioles. Such crystals have been noted also by Green (57) in mice of three strains, but apparently are not related to the occurrence of pulmonary tumors. Wells, Slye, and Holmes (201) added seven epidermoid tumors among 2865 mice with primary pulmonary neoplasms. Horn et al. (86) have described another type of pulmonary tumor in tiyo mice, in which a single layer of columnar cells containing mucus lined the pulmonary alveoli, with papillary tufted projections. The authors believed that these were cases of pulmonary adenomatosis in the mouse. An illustration published by Twort and Twort (195) in 1932 also presented a similar pulmonary tumor. Primary pulmonary tumors are usually seen in older mice, unless the neoplasms have been induced. The induced tumors seem to appear rather suddenly, grow to a size of 3 to 6 mm. in diameter, and then progress slowly, eventually coalescing with neighboring nodules. Relatively few mire dying with pulmonary tumors can be stated to have died of the neoplasm, although in some cases it might replace half of the thoracic space and invade the diaphragm or chest wall. Metastasis of pulmonary tumors is infrequent and late. Wells, Slye, and Holmes (201) found metastases in 104 of 2865 mice with pulmonary tumors, or 3.6%. All had metastases to the mediastinal lymph nodes, and ten to distant organs, including five to the kidney, three to the heart, and one each to the seminal vesicle and skull. One-third of the metastases were sarromatous in appearance, even when the primary neoplasm lacked sarcomatous elements. Campbell (32) found distant metastases in 3 of 192 mice with pulmonary tumors; two were to the kidney and one to the heart. In a group of 60 mice with pulmonary tumors induced by oral administrations of 1,2,5,6-dibenzanthracene,Magnus (132) found distant metastases in two, of which one was to the liver and one to a suprarenal gland. Thus, induced as well as spontaneous pulmonary tumors can develop distant metastases, in approximately 3 % of the cases. The monotonous adenomatous pattern, the uniformity in the size and shape of individual cells, and the infrequent mitotic figures in small pulmonary tumors suggest that the neoplasms undergo a ((benign” stage

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when they may be designated as adenomas. That all these tumors either originally or eventually are malignant is indicated by their lack of encapsulation, invasion of pulmonary tissues, progressive growth, metastasis, and transplantability to the subcutaneous tissue of genetically homologous mice. The terms that have been applied to these tumors include adenoma, adenocarcinoma, and papillary cystadenoma. Stewart (189) recently suggested “alveologenic carcinoma,” on the basis of histogenesis. It would appear to me that a purely descriptive and noncommittal name such as “primary adenomatous pulmonary tumor” for the common variety of these neoplasms still remains preferable. 2. Biochemical Properties

Biochemical comparisons between normal lungs and pulmonary tumors in mice are limited to the study of one transplantable pulmonary tumor. This is Andervont’s (4) Lung Tumor F, that arose in a strain A mouse and has maintained its adenomatous pattern for well over 100 passages. Greenstein (58) in his monograph summarized data on eleven comparable measures of enzymatic activity of this tumor and normal mouse lungs. The tumor was higher in xanthine dehydrogenase, and considerably lower in alkaline phosphatase and in esterase. The same range of activity as in normal lung was found in respect to arginase, acid phosphatase, ribonucleodepolymerase, deoxyribonucleodepolymerase, ribonucleodeaminase, and deoxyribonucleodeaminase. These characteristics are in each instance in conformity with findings on a wide variety of transplanted tumors in mice and in rats. The biotin content of Lung Tumor F is approximately half that of normal mouse lung (202). Schneider (164) reported that Lung Tumor F contained approximately three times the ribosenucleic acid and twice the deoxyribosenucleic acid content of normal lung tissue of mice. Thus, in biochemical terms, at least one transplantable pulmonary tumor has the general characteristics of malignant neoplasms.

V. HISTOGENESIS OF PULMONARY TUMORS IN MICE The earlier workers on pulmonary tumors in mice could not come to a conclusion regarding the histogenesis of the tumor. There was constant involvement of the alveoli, and no tumors appeared to be limited to the bronchioles, although they were sometimes involved by the neoplastic process. In general, the tumors were considered to arise from the alveolar cells or from the bronchial epithelium. The rapid production of pulmonary tumors in susceptible mice with carcinogenic hydrocarbons allowed a systematic investigation of their histogenesis by Grady and Stewart (56). Strain A mice were injected

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subcutaneously with 1,2,5,G-dibenzanthracene or 20-methylc holant hrene and killed a t frequent intervals, and a lobe of the lung was serially sectioned. C’onsecutive morphological changes in three dimensions could thus he follou-ed. Jlostofi and Larsen (141) recently repeated the study using urethane, and recorded a practically identical sequence of events. The initial morphological effect, seen during the first two weeks, is a proliferation of alveolar cells, particularly prominent in the subpleural alveoli remote from t hc bronchioles. .%mong the alveolar cells appear individual or small groups of enlargcd cells which sonietimcs project into the a\-eolar lumen. I h r i n g the third and fourth weeks there are islands of such cells, shon-ing increased numbers of mitotic figures, and by the sixth week these masses arc recognizable tumors. The bronchial epithelium shows no hyperplasia or proliferation, and there are no inflammatory changes. Only a t later stages are the tumors seen in close proximity or invading the lumen of the bronchioles. These studies coiiclusivdy demonstrated that the usual pulmonary tumors i n mice arise from what appear t o be the alveolar lining cells. Orr’s (148) opinion that pulmonary tumors in mice and in rats following urethane arise from bronchi within areas of inflammation and atalectasis appears to be erroneoub and attributable t o the examination of larger tumors at later stages of their growth. The nature of the cell from nhich pulmonary tumors in mice are derived is ohscure and relates to the general problem of the nature of the alveolar cell i n the lung. Whether these are epithelial cells or mesenchymal cells, or a combination of both, has heen the subject of considerable controversy obviously involving semantics as well as embryology. The reader is referred to papers by Bell (23) and by Geever ct al. (54) for further discussion. An interesting problem that is related t o the topic of the nature of the alveolar cell and tumors derived therefrom is the behavior of metastases and of transplants of the tumors. I t has already been rioted that one-third of metastases are sarciomatous i n appearance (201). Andervont (4),who i n 1937 first recorded the subcutaneous transplantation of pulmonary tumors, also obserl-ed that 3 of 7 such transplants changed into sarcomas in subsequent passages. The study was repeated (10) on 20 spontaneous and induced pulmonary tumors from three strains of mice, and 5 became sarcomas. Stewart, Grady, and Andervont (190) more recently reviewed and estended the investigation, using more precise histologic techniques, with exactly the same conclusions. The authors indicated a number of interesting postulations regarding the finding. It would seem t o this reviewer that two possibilities are most likely. One is that the alveolar lining cell is mesenchymal in origin and, on the one hand, possesses the

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property to appear epithelial and give rise to adenomatous epithelial-like tumors and, on the other, to revert to its mesenchymal nature and produce sarcomas. The other explanation is that the primary pulmonary tumors are mixed tumors containing both epithelial and mesenchymal elements. Breedis et al. (29),in a study of two transplanted pulmonary tumors, one of which altered in appearance on passage, concluded that this represented a modification in the appearance of the tumor cells and not an overgrowth of sarcomatous elements present in the original tumor. Newer methods of histochemistry and cytochemistry may yet yield additional information on these points.

TUMORS IN MICE The characteristic of different frequencies of pulmonary tumors in different strains of mice was appiied to genetic studies by Lynch (124-130) in 1926.Matings between a low-tumor strain 1194 of which 7 % developed pulmonary tumors, and a high-tumor strain Bagg albino, of which 40% developed pulmonary tumors, resulted in the F1 generation that resembled the high-tumor strain, and backcross generations resembled the strain to which the F, mice were mated. Lynch employed pulmonary tumors induced by tar-painting and injection of l12,5,6-dibenzanthracene as well as spontaneous tumors. Andervont (3, 6, S), using spontaneous and induced tumors, and Bittner (24,25) using spontaneous tumors in strains A and C57 black, obtained F1 and backcross ratios that also were compatible with an interpretation of single-dominant-factor inheritance of susceptibility. Extension of the work to crosses between other strains (11) indicated that although susceptibility was inherited in a dominant manner, multiple genetic factors or modifying factors had to be involved. Heston (69-73), and Heston and Deringer (78-82) have carried out extensive, exact studies on the genetic linkages between susceptibility to pulmonary tumors and known genes of mice. Most of the investigations have been performed with tumors induced by means of a single intrabut observations on venous dose of 0.5 mg. of 1,2,5,6-dibenzanthracene, spontaneous tumors thus far have reiterated the conclusions reached on induced neoplasms. The use of induced tumors not only allowed more rapid observations, but made it possible to consider latent time and number of tumors as well as the frequency of occurrence (Figs. 5 and 6). Outcrosses of strain A mice to three low-tumor strains showed that the response was different in each group, and indicated multiple genetic factors with cumulative effects. Linkage studies demonstrated an association between susceptibility to pulmonary tumors with a t least five genes. The lethal yellow gene (Au) increased susceptibility, although in the cross VI.

INFLUENCE OF HEREDITY IN PULMONARY

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FIG.5. Frequency of pulmonary tumors in strain A and strain L (C6, brown) mice and their F,. F,, and hackcross hybrids killed a t intervals following intravenous injection of 0.5 mg. 1,2,5,6-dibenzanthracene. Open circles represent groups of 20 to 108 mice; closed circles, groups of 15 to 18 mice. From Heston (71, 73).

:t

25

' 0) 0 L

,t

0-

STRAIN-L

.

-

STRAIN A

n

_cr__LG. Fl

251

25 50

t

b

A-BACKCROSS

L-BACKCROSS

0

20 40 60 80 Number of nodules

100

FIG.6. Frequency polygons for number of tumor nodules in strains A and L mice and their F1,F2,and backcross hybrids at 16 weeks following intravenous injection of 0.5 mg. 1,2,5,6-dibenzanthracene.From Heston (71, 73).

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employed it was introduced from the resistant strain. Flexed tail (f), hairless (hr), and the linked genes shaker-2 (sh-2) and waved 2 (wa-2) all were associated with a decrease in susceptibility. No association was found between susceptibility and eight genes, the leaden (In),piebald (s), dilution (d), agouti (a), brown ( b ) , waltzing ( v ) , waved-1 (wa-l), and pink-eye ( p ) genes. I n 1942, Heston (71) concluded that a minimum of four pairs of susceptibility genetic factors were involved in the difference between strains A and L, and that in the total variance in susceptibility 86% were due t o genetic factors and 14% to nongenetic factors. I n a later review (73) the opinion was modified t o stating that the number of genes involved was not known, and that it was not clear whether susceptibility is carried on the chromosomes with the marker genes or is an added effect of the identified genes. Later complete analysis (82) showed that the association between susceptibility to pulmonary tumors and the flexed tail and lethal yellow genes was attributable to the action of the genes per se. There was no association between these genes and susceptibility to mammary tumors or hepatomas. Burdette (30) studied the genic linkage of urethane-induced tumors, and found such association with shaker-2 and waved-2 genes, but not with the flexed-tail gene. This finding indicates that the linkage of susceptibility genes and certain marker genes might not be the same for tumors induced with urethane and with carcinogenic hydrocarbons. Additional complications are also introduced by the recent work of Deringer and Heston (43), in which it was shown that the Swiss (SWR) mice developed a greater mean number of pulmonary tumors than the strain A mice, and that in two crosses of strains the males developed more tumors than the females. Thus, it suggests that the number of nodules induced with intravenously injected 1,2,5,6-dibenzanthraceneis not necessarily controlled by factors identical with those controlling frequency of spontaneous tumors in all strains, and that factors connected with sex may exert an influence in certain crosses of strains. The reader is referred to the reviews of Heston (73, 75, 76) for a more extensive presentation of this line of investigation, and its relationship to heredity factors in other neoplasms.

HYDROCARBONS AND RELATED COMPOUNDS VII. POLYCYCLIC During the second decade of the century, several workers (45, 98, 200) recorded that pulmonary tumors were found in mice painted with tar. The interest was in cutaneous carcinomas, however, and the distinction between primary pulmonary tumors and metastatic deposits was not clearly made in the publications. Murphy and Sturm (144) in 1925 first

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hlICHhEL B . SHIMKIN

clearly demonstrated the carcinogenic effect of tar upon pulmonary tissue of mice by rotating the applications of tar over different areas of the skin and thus avoiding the appearance of ski11 cancers. Of 10 mice painted in this manner that survived for sis months, 85 % had multiple pulmonary tumors, whereas none of 38 untreated controls of the same stock had tumors. The possihility of the tar or some constituent thereof reaching the lungs was mentioned but discarded in favor of a postulation that tarpainting altered the body state so th at tumors occurred at points of incidental irritation. The findings of Murphy and Sturm (144) were confirmed and extended in a number of laboratories (27, 121, 157). Schabad (157-161) exposed mice to t a r by several routes, including subcutaneous injection, intravaginal and rectal introduction, and intraperitoneal injection, and elicited pulmonary tumors. The descendants of mice th a t developed pulmonary tumors were reported t o be more susceptible to pulmonary tumors than descendants of mice that did not; he postulated (158) th a t this was due to the transmission of the carcinogen to the progeny. \\.'ithin a few years after the publication of the report on the carcinogenic property of 1,2,5,fj-dibenzanthracene, a number of workers (1 11, 128, 159) found that a single subcutaneous injection of approximately 1 mg. of the chemical produced pulmonary tumors in mice. Andervont ( 2 ) reported that multiple tumors arose in 25 of 26 strain A mice within three months of the injection and before any had developed subcutaneous sarcomas, suggesting that the lungs of these mice mere a more delicate test object than the subcutaneous tissue. Preparation by Lorenz (118, 122) of carcinogenic hydrocarbon dispersions and adsorbed on charcoal allowed investigation of the effects of various routes of administration and of the physical state of the chemicals. Intravenous injection was the most effective route, producing the greatest number of tumors within a few weeks. Dibenzanthracene adsorbed on charcoal, and thus maintained at the site of injection, produced local sarcomas and few, if any, pulmonary tumors, 1vhere:ts the same preparation administered intravenously induced multiple tumors of the lung (15, 16). Contact of the lung with inserted threads coated with dibenzanthracene evoked not only the usual adenomatous humors but epidernioid carcinomas ill mice of strains A and ('3H (5, 9). In 1938, Andervont (7) published a study on the comparative susceptibility of eight inbred strains of mice t o the spontaneous occurrence and induction of pulmonary tumors following subcutaneous administration of 1,2,5,G-dibenzanthracene.Susceptibility t o induced tumors paralleled the spontaneous frequency of the tumors; i.e., mice most susceptible t o spontaneous tumors were also most susceptible to the induction of pul-

PULMONARY TUMORS I N EXPERIMENTAL ANIMALS

239

monary tumors with this carcinogen. Even mice of strains most resistant to spontaneous tumors, such as Cb7 black, developed some pulmonary tumors within 38 weeks following intravenous injection of 0.5 mg. of the carcinogen (10). Shimkin (170) extended the observations with intravenously injected 20-methylcholanthrene, with essentially identical

/ 8

14 Time in weeks

20

FIG. 7 . Response of lungs of strain A mice to a single intravenous injection of approximately 0.25 mg. of nine compounds dispersed in 0.25 cc. of water. Carcinogenic index is the per cent of mice with pulmonary tumors times the mean number of pulmonary tumors in positive animals. The broken line represents untreated control mice. A , 1,2,5,6-dibenzanthracene;B, 3,4,5,6-dibenzcarbazole; C, 3,4-benzpyrene; D, 15,lGbenzdehydrocholanthrene; E, 1,2,5,G-dibenzacridine; F, Zmethyl-3,l-benzphenanthrene; G, 4’-methyl-3,4-benzpyrene; H , 1,2-benzanthrscene; I, 3-methoxy-10propyl-1,2-benzanthracene.All but three points represent groups of 10 to 20 mice. From Andervont and Shimkin (17).

results. A detailed study (171) of the dose-time-response relationships of

the two carcinogens in strain A mice indicated that this was a rapid, sensitive, and reliable biological system for the quantitative testing of carcinogenic properties of a wide variety of chemical materials, as well as for other experimental applications. The procedure of pulmonary tumor induction as a test for carcinogenic properties of a number of chemicals was elaborated by Andervont and Shimkin (17). Figure 7 presents the results on nine compounds admin-

240

MICHAEL B. S H I M K I N

istered intravenously in single doses of approximately 0.25 mg. By consideration of the per cent of animals developing tumors and of the number of tumors at specific periods after injection, the quantitative carcinogenic potency of a wide range of chemicals for specific tissue can be derived, and most tests completed in four months. Perhaps the greatest disadvantage of the method is the lack of a clear end-point, since strain A mice develop pulmonary tumors spontaneously at an early age. Since all strains of mice develop the neoplasm if observed for a sufficient length of time, and since the susceptibility of the strains to spontaneous tumors parallels susceptibility to induction of such tumors, this objection cannot be overcome by the use of more resistant mice. Reliable conclusions can be derived only by having comparable control groups of sufficient number that are sacrificed at the same time as the experimental animals. With strain A mice, four months is a desirable time to kill the first groups of animals. It is more difficult to establish whether pulmonary tumors are produced in the course of an experiment in which the animals are permitted t o live out their life span. When the lungs of such mice are studded with multiple tumors, it is probable th at the tumors were induced. On this basis, 8.9-dimethyl-1 ,Zbenzanthracene, as reported by Shear (168), and several compounds included i n the tests of Badger et al. (20), of Dunlap and Warren (48), and of Law and Lewisohn (108) would be included as carcinogenic for the pulmonary tissue of mice. The investigations of Andervont and Shimkin (17) showed that there was no complete parallelism between the ability of some compounds to produce pulmonary tumors in strain A mice and their carcinogenicity as revealed by the induction of sarcomas following subcutaneous injection or of carcinomas following percutaneous applications. Badger et al. (20) and Iiennaway et al. (95) pointed out a number of polycyclic hydrocarbons that were of low but positive carcinogenic potency in producing cutaneous carcinomas in mice and were inactive in eliciting sarcomas upon subcutaneous injection. By the same token, no exact parallelism or even the same qualitative response should be anticipated in results obtained by the pulmonary-induction technique and in those obtained by the subcutaneous or percutaneous methods. The procedures are ancillary, and each contributes independent information. The statistical aspects of the reaction in strain A mice, including the number of animals to be considered in relation to the desired accuracy and levels of statistical significance, have been calculated by Shimkin and McClelland (179). Heston and Schneiderman (85) also presented statistical considerations of the response. Rogers (152), in using the size of nodules as well as their number, added another measure by which the actual mass of neoplastic tissue produced by carcinogenic stimuli

PULMONARY TUMORS I N EXPERIMENTAL ANIMALS

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can be determined with some degree of accuracy (Fig. 8). The application of quantitative differential techniques, such as devised by Chalkley (36), would make it feasible to determine the number of cells and their mass appearing at various periods under the stimulus of various doses of carcinogenic agents. The recent report of Telford and Jeffery (194a) is a step in this direction.

J

70

Mouse wt. Lung wt. 25

125

15

74

10

50

0.16

ail

‘hmope pep mouse

Total tumors

+at injection (weeks) FIG.8. The relationships between age, weight of lungs and body weight, and the induction of pulmonary tumors in Swiss mice with ethyl carbamate (urethane). Mice received single intraperitoneal injections of 1 mg. urethane per gram mouse and were killed seven weeks later. Each point represents the mean value of 25 animals. From Rogers (152).

No specific reports have been noted on the induction of pulmonary tumors in rats with carcinogenic polycyclic hydrocarbons. Even such an extensive investigation as that of Dunning, Curtis, and Bullock (49), who injected subcutaneously 1,2,5,6-dibenzanthraceneand 3,4-benzypyrene into 688 rats of six strains, made no mention of the presence or absence of pulmonary tumors, but the paper reveals that the interest was limited to the subcutaneous neoplasms. That pulmonary tumors can be induced in rats following such procedure is indicated by Lewis and King (112), who comment upon the presence of “lung neoplasms’’ in the King strain of

242

M1CH.IEL B . SHIMKIN

rats following the sucutaneous injection of 1 t o 4 mg. of dibenzantkracene, benzpyrene, or methylcholanthrene. Apparently no pulmonary masses were seen in 14 other strains used by the authors, and the lesions in the King strain are not descrihed or illustrated. Following the intravenous injection of 5 mg. of methylcholanthrene into a few rats of the Wistar, C'olumbia, and Buffalo strains, Shimkin (172) obtained a single pulmonary tumor in one of five Buffalo rats a t 6 months, and definite multiple pulmonary tumors in another Buffalo rat killed a t l l months. These tumors were histologically indist iiiguishable from the adenomatous pulmonary tumor of the mouse. In guinea pigs no pulmonary tumors were observed following subcutaneous injection of 20 t o 40 mg. of methylcholanthrene, although subcwtaneouc; sarcomas were elicited in 29 of 34 animals (180). Heston and Deringer ( S l ) , howevcr, injected 10 t o 30 mg. of methylcholanthrene intravenously and obtained pulmonary tumors in 17 of 51 guinea pigs of strain 2 and in 13 of GO animals of strain 13. These neoplasms appeared t o be of thr same histologic type as the mouse tumors and were found t o occur in greater multiplicity among female than among male atiirnals.

YIII. VKETH ISE A N D R E L ~ T ECOMPOUNDS D In the course of in\wtigating the effects of exposures t o roentgen rays. Srttleship, Henshan., and JIeyer (145) in 1043 encountered unexpected multiple pulmonary tumors in their experimental mice of strain C'J-I. -Analysis of the observation and futher studies established that thc anesthetic used. ethyl carbamate (urethane), was the causative agent. IIenshaw arid Meyer (68) reported that singlc intraperitoneal doses of 1 mg. of iirethane per gram body weight in strain A mice produced an average of 9.5 pulmonary tumors in all of 18 mirr hy four months, and the response was linearly increased t o an average of over 30 modules when four or five weekly injections \\-ere given. Subcutaneous injection and oral aclniinistratioiis also produced pulmoiiary tumors, but no other neoplastic reactions (67, 166). I t was thus established that urethane was a potent carcinogen for the pulmonary tissue of mice, and apparently did not produce neoplasms a t the site of injection or other distant sites. There \\-as immediate and wide interest in the observations, since urethane is an old, well-known chemical, of simple molecular structure, water-soluble, and obviously more applicable to, and convenient in, many studies than the carcinogenic hydrocarbons. The morphology and histogenesis of the pulmonary tumors are identical with spontaneous neoplasms or those induced with the hydrocarbons. The response of mice t o pulmonary tumor induction with urethane is parallel t o their suscepti-

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243

bility t o spontaneous neoplasms of the lung (40), although Shapiro and Kirschbaum (167) reported that the NH strain of mice may be an exception to this conclusion. Larsen (100-104) carried out a series of systematic studies on the relationship of chemical structure and biologic activity of urethane and related materials. He showed that the anesthetic action of urethane was not involved in its ability to evoke pulmonary tumors (100). Twelve barbituric acid derivatives and nine miscellaneous hypnotics including paraldehyde, ethanol, and chloral hydrate did not produce pulmonary tumors in strain A mice. Orr (147) also found th a t three hypnotic agents, including nembutal, were negative for pulmonary carcinogenic activity. An examination by Larsen (102) of various esters of carbamic acid showed ethyl carbamate t o be a t least 20 times more active than any of the esters tested. Isopropyl, n-propyl, and trichloroethyl carbamates had some activity, whereas methyl, n-butyl, isoamyl, and chloroethyl esters were negative. Study of nitrogen-alkylated derivatives of ethyl carbamate demonstrated (103) th at n-isopropyl urethane, and methylene and ethylidene duirethanes a t 0.5 mg. per gram mouse per week for 13 weeks were active, but less so than ethyl carbamate. This suggested that the carcinogenic activity of alkylated urethanes may have been due to preliminary dealkylation t o ethyl carbamate. Possible degradation products of urethane, including ammonium carbamate, sodium bicarbonate, ammonium chloride, and potassium cyanate, had no effect upon the frequency of pulmonary tumors in strain A mice (104). The effects of urethane were explored in other species. No pulmonary tumors were obtained by Cowen (42) in guinea pigs or in chickens. Gross et al. (61) reported that white-footed deer mice (Peromyscus leucopus noueboracensis) were completely resistant t o lung tumors following 16 weekly intraperitoneal injections of urethane. Jafft5 and JaffB (91, 92) first recorded th a t pulmonary tumors could be induced with urethane in rats. Rats were maintained on 0.15% urethane in the diet, or received 30 intraperitoneal injections of approximately 100 mg. each during three months. Of 38 animals surviving for one year, 8 had primary pulmonary tumors. Four hepatomas were also found, and the authors believed they were induced by urethane-a point that requires confirmation. Guyer and Claus (62) administered three to five intraperitoneal injections of 1 cc. of a 10% solution of urethane, and elicited pulmonary tumors in 66 of 91 rats within eight to ten months. Mostofi and Larsen (140) also produced pulmonary tumors with urethane in the Wistar strain of rats. The histologic appearance and the histogenesis of pulmonary tumors in rats are apparently identical to the tumors evoked in mouse. It should be noted, however, that metastasis and trans-

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MICHAEL B . SHIMKIN

plantation of the rat tumors still remain t o be described.' The relative susceptibility of various strains of rats t o urethane-induced neoplasms also has not been investigated. The addition of the rat to experimental animals in which pulmonary tumors can be readily induced is of obvious importance and makes feasible a number of physiological and biochemical approaches that would be much more difficult to carry out on the mouse.

Ix. O T H E R

C H E M I C A L A N D P H Y S I C A L AGENTS, INCLUDING IKH.~LANTS

1. Miscellaneous Agents

hlorosenskaya (137, 138) and -4ndervont (10) established th a t o-amino-5-azotoluene, when injected subcutaneously or intravenously or administered in the diet, significantly raised the frequency of pulmonary tumors in susceptible mice. The agent also induced hepatomas and hemangioendotheliomas, and the chief interest has been directed toward these neoplastic reactions. The dose is greater than necessary with polycyclic hydrocarbons, 10 to 100 mg. usually being given over a period of some weeks. I n the extensive investigations of carcinogenic azo dyes in rats, no mention is made of the presence of primary pulmonary tumors, suggesting that the compounds are not carcinogenic for the pulmonary tissue of the rat. Bielschowsky (23) reported the induction of pulmonary tumors in 11 of 104 rats with 2 amino- and 2-acetylaminofluorene. The agents were given in the diet, about 4 mg. per day for 30 weeks. The pulmonary tumors that were produced were apparently of the same adenomatous type as seen following the administration of urethane, although squamous metaplasia of the bronchi appeared t o be more common (149). The great number of other neoplastic reactions th a t is evoked by the fluorenes obscures the relatively weak effect th a t is demonstrated in the lung. Morris et al. (139) reported no pulmonary tumors in rats of the Minnesota, Wistar, and Buffalo strains maintained on diets containing 2-nitro-2-amino-2-acetyl- and 2-diacetyl aminofluorene, although two rats developed lymphosarcoma of the lung. -4 progress report by Harding and Green (64) stated that multiple pulmonary tumors were induced in cats that mere maintained on diets containing 2-acetyl aminofluorene, but no complete report of this interesting finding has become available. Law (107) in a single study injected sodium deoxycholate and cholic 1 A urethsne-induced pulmonary tumor of the M-520 strain rat was successfully transplanted by Larsen in 1951 and is being maintained at the National Cancer Institute (187a).

PULMONARY TUMORS I N EXPERIMENTAL ANIMALS

245

acid intravenously in strain A mice. Of 20 mice that received deoxycholate, 16 developed pulmonary tumors by six months, a t a time when 18 of 38 untreated controls had such tumors. The difference is significant, but the experiment bears repetition before it is concluded that sodium deoxycholate is carcinogenic for the pulmonary tissue of mice. Heston (74, 77) has shown that the nitrogen and sulfur mustards are carcinogenic for the pulmonary tissue of mice. Two to four intravenous injections of approximately 0.025 mg. of methyl bis (0-chloroethyl) amine hydrophloride (HN,) produced an average of 3.5 pulmonary tumors in all strain A mice in four months. Positive effects were also obtained by the intravenous injection and inhalation (77) of bis (0-chloroethyl) sulfide. Nitrogen mustard, as urethane, is used in the clinical management of leukemia and lymphoma. Shimkin et aZ. (175) reported that a related compound, trisethylene-imino-s-triazine (TEM) also produced pulmonary tumors following one or two intraperitoneal doses of 0.05 mg. in strain A mice. An investigation (174) of other agents used clinically for the lymphomas was negative ; this included potassium arsenite, 1,4-dimethylsulfonyl butane (myleran), 4,4’diamidinostilbene (stilbamidine), 4-aminopteroylglutamic acid (aminopterin), and 17hydroxy-1 l-dehydrocorticosterone (cortisone). The effect of ionizing radiations upon pulmonary carcinogenesis in mice was investigated by Lorenz et al. (120). Mice of strain A were exposed to total body irradiation from a radium source for 8 hours daily for nine months, receiving a total dose of approximately 2500 ry. Of 43 control animals, 47 % had pulmonary tumors, whereas among 55 irradiated mice 77 % had tumors and a higher proportion of these were multiple. The difference is statistically significant, but certainly the effect is not marked, and apparently quite slow in comparison with such agents at the carcinogenic hydrocarbons, urethane, or nitrogen mustard. An interesting question remains as to whether the effect is mediated through the same mechanisms. Heston and Lorenz (84) indicate that the combination of exposure to x-ray and nitrogen mustard does not enhance the induction of pulmonary tumors, at least not under the conditions of their experiment. This might suggest the possibility of different modes of action for the two agents, although further work is necessary to establish the point. Intravenous injection of colloidal thorium dioxide did not increase the number of pulmonary tumors in strain A mice (18). Lorenz et al. (120a) found 18 pulmonary tumors, of which 10 were multiple, in 42 guinea pigs exposed to long-continued total body gamma irradiation that survived for three to six years. Among 19 unexposed animals, 3 had single pulmonary tumors.

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MICHAEL B. SHIMRIN

A detailed analysis was made hy Blum (26) of the effect of ultraviolet radiation upon the frequency of pulmonary tumors in strain A mice, a high proportion of which developed skin carcinoma and subcutaneous sarcoma. Xot only was there no increase in the frequency of pulmonary tumors, but actually the experimental mice had somewhat fewer tumors. Thus, this form of carcinogenic modality is apparently localized to the site of contact, and its carcinogenic effect is prohnbly not mediated through the formation of hydrocarbon-like carcinogens from body constituents such as rholesterol. Copeland and Salmon (38, 50) have published interesting observations on the development of neoplasms i n rats on prolonged diets deficient in choline. I t wis reported that 385; of the rats developed pulmonary tumors, and 3 0 5 had aderiocarcinoma of thc liver. The neoplastic nature of the pulmonary lesions has bccn questioned, and the finding is considered t o be sub jitdice a t this time. The wide variety of chemical and physical agents that have been shown t o produce pulmonary tumors i n mice and rats provides an irrefutable demonstration of the relative nature of the concept of carcinogenicity. Table I1 presents the observations on six types of carcinogenic T;IBLE I1 Carcinogenic Properties of Six Carcinogens upon Four Tissues in MiceY

Carcinogen 20-~lethylcholanthrenc If, 10-l)iitiethylanthrncerie 3,~,5,C-I~ihmzcarhazole o-Amino-5-azotoliiene Ethyl ralhnniatr Ultra\ iolet rad13tion

Cutaneous Carcinoma

++ + ++ -

++

Pulmonary Suhcuta~ieous Adenomatous Sarcoma Hepatoina Tumor

++ ++ ++ -

-

++

+-I-

++ + ++ + ++-

0 I’lrrs signs indicate the iniluctiun of neoplasnrs; iuinus signs indicate that such tuniors have not been elicitcd.

stimuli in mice. On the one hand, there are chemicals such as 3,4,5,6dibenzcarbazole, which induces cutaueous carcinoma and subcutaneous sarcoma a t the site of application, and hepatomas and pulmonary tumors a t distant sites. I-ltraviolet radiation, on the other hand, produces tumors a t the site of contact in the skin or immediate subcutaneous tissue, and has 110 distant carcinogenic effects. Aminoazotoluene produces hepatomas, pulmonary tumors, hemangioendotheliomas and sarcomas in mice, and hepatomas in rats. These tremendous differences in effects,

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relative t o species, tissue, and type of neoplasm induced, should certainly indicate that an over-all explanation of carcinogenic reaction is hardly to be anticipated. 2. Exposure to Inhalants I n attempting to demonstrate that inhalation of environmental dusts and fumes might have a causal connection with the development of pulmonary carcinoma in man, a number of investigations have been performed on the effect of such inhalants in animals, particularly mice. As far back as 1923, Kimura (96) claimed that one adenocarcinoma was produced in a guinea pig following the intratracheal introduction of coal tar, but his description and illustration are not convincing. Valade (197) failed t o elicit tumors in rats by the intratracheal injection of methylcholanthrene. An increase in pulmonary tumors in strain A mice by the intratracheal introduction of methylcholanthrene was produced by Shimkin (169). R. E. Smith (184), in 1928, kept mice for 6 hours daily and then three times a week in atmospheres containing coal tar fumes and fumes from an automobile engine. No increase in pulmonary tumors was observed. Campbell (31-35), in an extensive series of investigations from 1934 to 1943, exposed mice to various dusts over protracted periods. I n 100 mice exposed t o dust from tarred roads, 71 developed pulmonary tumors and 59 developed skin carcinomas, as compared with 90 controls, of which 7 had pulmonary tumors and no skin cancers. Exposure of mice t o motor exhaust gas, carbon monoxide, and cigarette smoke did not result in significantly greater frequency of pulmonary tumors. Dusts from various sources were also examined, and a number of them increased the frequency of pulmonary tumors. In the summary article, Campbell (35) reluctantly concluded that these effects were of a ‘ I prolonged chemical nature, although it is not possible a t the moment t o exclude entirely some effect of prolonged mechanical irritation.l 1 Seelig and Benignus (165) performed a similar experiment, exposing mice t o soot from coal smoke, and obtained eight tumors of the lung among 100 Buffalo strain mice, whereas one tumor was found in 50 controls. McDonald and Woodhouse (135) also increased the frequency of pulmonary tumors in mice that inhaled dust which on spectrographic analysis contained benzpyrene. Leiter et a2. (109, 110) showed that benzene extracts of atmospheric dusts gathered in a number of cities in the United States produced sarcomas at the site of injection in 30 of 372 mice. These studies demonstrate that dusts and other atmospheric contaminants that may be expected to contain chemical agents of the polycyclic hydrocarbon type, are carcinogenic for the pulmonary tissue

248

MICHAEL B. SHIMKIN

of mice when introduced as inhalants, and for the cutaneous and subcutaneous tissues of mice upon suitable application. Studies with tobacco fumes are somewhat more controversial. Loren2 et al. (123) failed t o elicit pulmonary tumors in strain A mice exposed to tobacco fumes. Essenberg (51) , however, succeeded in demonstrating that cigarette fumes increased the number of such tumors in mice. Strain A mice were exposed t o cigarette fumes for one year, and 21 of 23 animals developed pulmonary tumors, as compared with a frequency of 59 % among 32 unexposed controls. In this connection, the carcinogenic effects of the products of tobacco has been described by Koffo (153), who obtained carcinomas of the ears of rabbits painted with tars from tobacco, and by Flory (53), who obtained papillomas and two carcinomas in mice. The recent publication of Wynder el a2. (209) summarizes the subject of experimental production of carcinoma in mice with cigarette tar, and reports the induction of epidermoid carcinoma in 44% of 81 mice painted with condensates obtained from cigarettes. The evidence seems convincing that under certain conditions tobacco fumes as well as coal fumes may contain carcinogenic materials. In a short abstract] Lisco and Finkel (114) mention th a t rats exposed to inhalations of radioactive cerium developed metaplastic changes in the bronchial epithelium and malignant tumors arising therefrom. Completc descriptions would be useful, particularly since epidermoid carcinoma of the lung has been produced but rarely ( 5 , 88) in experimental animals. r'orwald (I 99) has reported the appearance of pulmonary tumors in rats exposed t o inhalations of beryllium sulfate aerosol.

X. FACTORS AFFECTING PULMONARY TUMOR INDUCTION I N MICE 1. Sex, Age, Diet, and Inflammation

It has already been noted that pulmonary tumors in mice, either of spontaneous occurrence or induced with various carcinogens, appear t o be less dependent upon and less affected by different physiological states and environmental factors than many other types of neoplastic growth, particularly the mammary carcinoma and the hepatoma. The orcurrence of pulmonary tumors and their induction are the same in the male arid the female mice, although some differences have been reported in crosses between inbred strains (43). T h a t the tumor is independent of hormonal factors is also indicated by the lack of effect of breeding, of endogenous injections of estrogens or androgens, of deoxycorticosterone (176), or of cortisone ( 1 7 4 . Foster-nursing, i.e., the presence or absence of the Bittner milk agent necessary for the appearance of most mammary tumors in mice, has no effect upon the frequency of pulmonary tumors (21, 178).

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The investigations on carcinogenesis in embryonic lungs and in young mice (152, 185, 189) prove that these rapidly growing tissues are more susceptible t o the effects of carcinogens than lungs of adult mice. Age after maturity, however, does not have a definite role, since little if any difference in response is seen in mice that are between 2 months and 11 months in age (152, 178). Restriction in diet is one of the few environmental conditions that has been demonstrated t o affect the occurrence of pulmonary tumors in mice. Tannenbaum (193, 194), in his systematic study of the subject, showed that the frequency of spontaneous pulmonary tumors was reduced by approximately 50% in Swiss mice on restricted diets. A decrease in the frequency of pulmonary tumors by some 30% by underfeeding was also demonstrated in strain A mice by Larsen and Heston (105). This appears t o be an over-all effect related t o caloric restriction and body weight, and is not attributable to more specific restrictions in fat, carb ohydr at es, or cystine. Special mention must be made of the role of nonspecific chronic or acute inflammation and irritation upon the initiation of pulmonary tumors in mice. Many colonies of laboratory mice are plagued by the constant or intermittent occurrence of parasites and other infections, and it is not surprising that connections are sometimes drawn between such infections and neoplasia. The early morphologists soon discarded such postulations, and studies on the histogenesis of pulmonary tumors definitely fail to connect any morphological evidence of inflammation with the initiation of the tumors. An investigation was carried out by Shimkin and Leiter (177) which further dissociates inflammation from the spontaneous or the induced neoplastic reaction. Strain A mice were injected intravenously with finely ground arsenopyrite, chromite, thorite, or quartz. Despite the presence of the ore particles in the lungs and histologic evidence of chronic irritation, the frequency of pulmonary tumors was not increased. The injection of 0.1 mg. of methylcholanthrene concurrently with the ores resulted in no increase in the number of induced nodules as compared with mice that received only the carcinogen (Fig. 9). Soot from a chimney burning soft coal, a benzene extract of which induced subcutaneous sarcomas in C,H mice, increased the frequency of pulmonary tumors. Lorenz and Andervont (119) raised mice in special dust-free chambers in which the atmosphere contained only 3% of the dust that was found in the surrounding laboratory. This reduction in atmospheric dust did not influence the development of pulmonary tumors when the mice were injected subcutaneously with 1,2,5,6-dibenzanthracene. Steiner and Loosli (188) infected mice with influenza Type A. Proliferation of the bronchial epithelial cells typical of the infection was observed,

250

MICHAEL B. SHIMKIN

but the surviving mice did not shorn any increase in the frequency of pulmonary tumors. I n fact, there were only 20 such tumors among 250 virus-infected strain A mice, whereas among 105 controls, 38 pulmonary tumors were found-a significantly higher frequency. No pulmonary tumors were elicited in the C57 black mice. Glover and Jennings (Ma) showed t ha t infection of mice with the grey-lung virus did not affect the

10

3

4.5 Time in months

6

FIG.9. Response of lungs of strain A mice to intravenously injected ores and soot. A , single injection of 5.0 mg. of arsenopyrite, chromite, or thorite, or 1.0 mg. of quartz. R , untreated controls. C , single injection of 2.5 mg. of soot. D, injection of ores as in A, 0.1 mg. methylcholanthrene a week later. E , single injection of 0.1 mg. methylcholanthrene. Carcinogenic index is the per cent of mice developing pulmonary tumors times the mean number of tumors per positive animal. Each point represents from 8 to 60 mice. From Shinikin and Leiter (177).

production of pulmonary tumors in mice with urethane. “Reticuloendothelial blockade” by the injection of trypan blue had no effect upon the induction of pulmonary tumors in mice (178). The available evidence clearly dissociates nonspecific irritation and inflammation from the occurrence of pulmonary tumors in mice. The dissociation seems to hold also, on histologic evidence, in pulmonary tumors induced in rats with urethane (140, 154) and in guinea pigs with intravenous methylcholanthrene (81). Whether this holds for human beings or any other species should not be adduced without reservation. The increase of pulmonary tumors in mice following exposure, inhalation, or injection with a material is good evidence of a presence of a carcino-

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genic agent in the material, an agent carcinogenic for the pulmonary tissue of mice. 2. Pulmonary Tumors in Embryo Mice

Law (106) in 1940 injected 0.125 mg. of 1,2,5,6-dibenzanthraceneinto the aminotic fluid of mice three to six days pre-partum, into mice 24 hours old, and into two-month old mice. Six to eight months later, 80% of the mice which were exposed to the carcinogen in utero, and 100% of those injected within a day after birth had multiple pulmonary nodules, whereas of 29 animals injected at the age of two months only 2 had tumors. This proved that embryonic lung was responsive to the carcinogen and suggested that it was more susceptible than adult lung. Larsen (101) and Klein (97) reported a high frequency of pulmonary tumors in the offspring of mice injected intravenously with urethane during pregnancy, especially if the urethane was administered during the last day of parturition. This established the transplacental effect of the compound, and that the penetration was increased just before parturition. W. E. Smith (185), by the ingenious method of exposing strain C mouse embryo lungs to methylcholanthrene in vitro and then transplanting them to the subcutaneous tissue of adult strain C mice, was able to produce adenocarcinomas at the site of implantation within three weeks. Since pulmonary tumors in adult strain C mice following 0.5 mg. of methylcholanthrene arise between 13 and 20 weeks, and certainly not as early as 6 weeks, the observation suggested that embryonic lung was more susceptible to carcinogenesis than adult tissue. Studies on the injection of pregnant strain C mice with urethane revealed that adenomatous growths were present in the offspring three and ten days old (187). Thus, the carcinogenic reaction was shown to occur much faster in the embryonic lungs than in adult tissue. It is of interest that mitoses were abundant in the tumors thus induced for the first two months, after which cell division almost ceased, indicating that the carcinogenic reaction to urethane, as t o methylcholanthrene, is a self-limited one. Rogers (152) recently extended the studies in Swiss mice. He found that urethane, given in doses per weight of the animals, produced more pulmonary tumors in rapidly growing mice, up to six weeks in age, than in adult mice. Urethane injected into mice who already had pulmonary tumors induced with previous treatment with urethane produced an increased number of nodules, but the size of the nodules was not increased, indicating that the role of urethane was to initiate neoplastic change and that urethane did not lead neoplastic cells to multiply. In this connection, colchicine had no effect upon the frequency of spontaneous pulmonary tumors, nor upon the induction of such tumors with urethane.

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MICHAEL B. SHIMKIN

XI. krECHANISM OF INDUCTION OF PULMONARY TUMORS I N MICE The mechanism of induction of pulmonary tumors in mice can be divided into two main considerations: (1) the mode of action of the carcinogen; and (2) the mode of response of the organism or tissue to the stimulus. 1 . Mode of Action of the Carcinogen The appearance of pulmonary tumors in mice injected with carcinogenic hydrocarbons and other carcinogens can be postulated to be due either to the local action of the carcinogens on the pulmonary tissue, to a general systemic action of the compound resulting in a lowered resistance to tumor development, or to a combination of the two. With the polycyclic hydrocarbons, the most efficacious method of inducing primary pulmonary tumors in mice, both from the standpoint of the shortest latent period and the greatest response as measured by the number of discrete tumors, is by intravenous injection. This route produces the maximum contact of the carcinogen with the lungs. Moreover, more lung tumors are produced by dispersions containing larger particles, of which 607, become lodged in the lung, than with smaller particles, of which only 10% are retained in the lung (178). This shows that the number of pulmonary tumors induced in strain A mice is a function of the dose of the hydrocarbon that reaches and lodges in the lungs, rather than of the dose injected into the whole organism. Pulmonary tumors are also induced when threads coated with 1,2,5,6dibenzanthracene are placed through the lungs of mice, or when dispersions of the compound are introduced into the trachea. Experiments with charcoal-adsorbed dibenzanthracene also yield important support to the belief that actual contact of the carcinogen and the lung is necessary for the neoplastic reaction. In general, more pulmonary tumors are evoked when a carcinogen is injected in media that leave the site of injection than in menstrua that are retained at the site of injection. After subcutaneous or oral administration of large amounts of 1,2,5,6dibenzanthracene, absorption spectrum analysis fails to reveal the presence of the compound or of its derivatives in the lungs, although multiple pulmonary tumors are induced. That the agent or some derivatives reach the lungs is suggested by the presence of photodynamic activity in suspensions of lungs of mice injected subcutaneously with 3,4-benzpyrene (142). With 1,2,5,6-dibenzanthracenelabeled with radioactive carbon a t positions 9 and 10, and injected intravenously as an aqueous colloid in doses of 0.5 mg., Heidelberger and Jones (65) recovered only 0.4% of the dose in the lungs & hour and 24 hours later. Less than 1% of 0.45 mg. of 3,4-benzpyrene labeled at the 5 position was recovered in the lungs (66).

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No radioactivity could be detected in the respiratory carbon dioxide. These findings are in strange contrast, perhaps explainable on the basis of the size of the particles in the dispersions, with the recovery of 50% of 0.5 mg. of 20-methylcholanthrene from the lungs of mice 30 minutes following intravenous injection-the detection being accomplished by absorption spectrum analysis of Lorenz and Shimkin (121). The amounts of the carcinogen in the lung decreased to one-tenth in four days, and extracts of lungs three days after injection induced sarcomas a t the site of subcutaneous injection in recipient mice, confirming biologically the presence of a carcinogen. The distribution and elimination of urethane labeled with radiocarbon a t the carbonyl and the ethoxy positions has been published by Skipper et al. (182). After an equilibration of the injected material during the first 30 minutes, approximately 7 % of the activity is excreted per hour via the respiratory route. At 24 hours, a t least 97% of the administered dose can be accounted for in the’expired air. Malmgren and Saxen (133) also demonstrated biologically that the carcinogenic effect of urethane is confined to the first 24 hours after injection. All evidence would appear t o indicate that the carcinogenic reaction in the induction of pulmonary tumors is the result of a direct contact of the carcinogen with the lung. The reaction is modified by the dose of the carcinogen, the route of administration, and the physical state of the agent. With the polycyclic hydrocarbons, urethane, nitrogen mustard, or trisethylene melamine, one injection is sufficient to evoke the neoplastic reaction. Whether this applied to roentgen rays and other stimuli remains t o be established. The division of the dose of methylcholanthrene (178) or of urethane (152) over a period of several days evokes no more pulmonary tumors than the same dose administered in a single injection. This would suggest that the reaction is an acute one and that prolonged exposure of the animal or of the tissue to the carcinogenic agent is not required. The reaction, once invoked, apparently proceeds without the necessity of the presence of the initial stimulus. Studies (179) on the quantitative aspects of dose-time response to methylcholanthrene have yielded some additional information. Gross observations of induced pulmonary tumors suggested that they appeared suddenly, grew rapidly for a number of weeks, and then increased only slowly in size. Figure 10 shows that the number of nodules produced a t various intravenous doses of methylcholanthrene increased markedly between the eighth and thirteenth weeks, and hardly a t all during the subsequent five weeks. The reaction is thus apparently a self-limited one. Rogers (152) also reached a similar conclusion in his fine studies on urethane-induced tumors in mice.

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0.0625 0.125 0.25 0.5 1.0 Methylcholanthrene in milligrams FIG. 10. Dose-time-response relationships to strain A mice injected intravenously with methylcholanthrene. The mean number of pulmonary nodules per mouse is plotted against dose on a log-log scale. Each point is represented by groups of 25 to 30 mice. The response a t 8, 13, and 18 weeks after injection is indicated, showing the increase in number between 8 and 13 weeks, and practically no increase between 13 and 18 weeks. Data from Shimkin and McClelland (179).

2. Mode of Reaction of the Lung The susceptibility of mice of various homozygous strains t o induced pulmonary tumors is parallel to the susceptibility of the strains t o the spontaneous development of this neoplasm. This suggests th a t the carcinogens, whether they be polycylic hydrocarbons or urethane, are accelerators of some process inherent in the animal. T ha t the locus of the reaction and that the susceptibility factors are in the lungs and not mediated through physiological changes in the whole orgallism has been demonstrated beautifully in two studies. Smith (185) and Horning (88) have shown th at carcinogenesis can be induced in tissues exposed t o carcinogens in vitro and then transplanted subcutaneously into compatible animals. Shapiro and Kirschbaum (167) obtained F1mice from a cross between Bagg albino, a pulmonary-tumor-susceptible strain, and dba, a pulmonary-tumor-resistant strain. Transplants of lungs from one-day-old mice from either parent strains into the subcutaneous tissue of the ear

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were shown to survive in these hybrids. In mice implanted with lung tissue from Bagg albino, and then injected intraperitoneally with urethane, pulmonary tumors were produced in 12 of 17 transplants. When the transplanted lung was derived from dba mice, only one pulmonary tumor was produced in 17 transplants. Heston and Dunn (83) transplanted lungs of a resistant strain (CU leaden or L) on one side and lungs of a susceptible strain (A) into the other flank of LAF, mice, who were then injected intravenously with l72,5,6-dibenxanthracene.Pulmonary tumors developed in 40% of the transplants from strain A donors and in 4% of transplants from strain L. What occurs in the lungs when the carcinogen reaches the susceptible tissue and what determines the differences in susceptibility to the neoplastic reaction in mice of different genetic backgrounds are questions that remain as unanswered for pulmonary tumors as for any other type of neoplastic transformation. As such, all theories that may be applicable to other forms of neoplasia are also relevant to pulmonary tumors. Analogies to the localization of intravenously injected Shope papilloma virus in the tarred ears of rabbits can be drawn, but no evidence exists that a viral agent is involved in the pulmonary tumors of mice, or that nonspecific chronic irritation in the lungs of mice enhances the neoplastic reaction. From quantitative dose-response relationships Heston and Schneiderman (85) have suggested that the reaction is a single, one-step change in the cell. As the authors carefully state, if this were a genic change it would be assumed to be a dominant mutation. The evidence is obviously insufficient to establish that the locus of reaction is a gene, and the term “somatic mutation” has validity only if it is used in the general sense of a change rather than in its more precise genetic meaning. Except for the studies on the decrease in frequency of pulmonary tumors in mice maintained on restricted diets, relatively little attention has been directed toward influences that may inhibit the appearance of the tumors. It has been noted that mice surviving infection with influenza had fewer pulmonary tumors than the normal controls (188), and that mice developing cutaneous carcinoma following ultraviolet irradiation had fewer pulmonary tumors than similar mice that did not develop skin tumors (26). Cowen (41) reported that the induction of pulmonary tumors with urethane was inhibited by injecting the mice with pentose nucleotides. Rogers (152a) also stated that intraperitoneal injection of a deoxyribonucleate inhibited the formation of pulmonary tumors with urethane. Only one attempt was found to follow specific biochemical changes in the lung as it undergoes carcinogenesis. Alkaline phosphatase was studied by Greenstein and Shimkin (59) in the lungs of strain A mice following

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intravenous injection of methylcholanthrene. The phosphatase activity remained completely normal, although the tumors themselves showed a decrease t o about 25% as compared with normal lung. Thus, there was either an abrupt change, or the chemical analyses were not sufficiently sensitive to reveal subtle changes in the tissues. A study was carried out by Lorenz and Shimkin (121) on the elimination of methylcholanthrene from the lungs and bodies of strain A and strain Cb7black mice, postulating that the difference in susceptibility to the carcinogen may be reflected in the ability of the animals to conjugate, detoxify, and eliminate the chemical, but no differences in the rates were observed. It would be of interest to determine whether a protein-carcinogen binding occurs in the lungs of mice and whether it is different in strains of different susceptibilities, analogous to the important studies on the azo-liver relationships reported by Miller and Miller (136). At the present time, the conclusions must still be restricted to the ones reached by Shimkin and Lorenz (178) in 1942: (1) carcinogens act directly upon the pulmonary tissue, (2) the pulmonary tissue is the locus of a process, present in varying degrees in all strains, that under normal conditions eventuates in the development of spontaneous tumors; (3) the carcinogen accelerates this process, both in regard to time of appearance of the tumors and in the number of discrete sites of carcinogenesis, and (4) the acceleration of the process is rapid and apparently the increased pace continues without further stimulation by the chemical agent. XII.

PULMON.4RY

TUMORS IN

M A N .4ND

GENERALDISCUSSION

I . Bronchogenic Carcinoma

The great majority of primary epithelial tumors of the lung in man are bronchogenic carcinomas, which may be undifferentiated, adenocarcinomatous, or epidermoid in pattern. There seems to be little question but that practically all arise from the basal cell layer of the bronchi, and some three-fourths from the epithelium of the larger bronchi (113, 203). It is one of the more malignant of the neoplasms of man, as manifested by its rapid invasion of surrounding tissues, its early, wide metastatic spread which may involve almost any organ or tissue, and its ability to kill the host within a relatively short period after the diagnosis has been established. The neoplasm is usually single and occurs several times more frequently among males than among females. The marked increase in frequency of bronchogenic carcinoma among males in the United States and in Europe during the past 30 years has created a problem of great interest and concern to clinicians and public health officers. The evidence (37, 94, 99, 206) is unequivocal and cannot

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be explained by changes in the age of the population, diagnostic improvements, or statistical inaccuracy. Epidemiological studies (192a) that have been carried out in the United States, England, Denmark, and Holland now allow a number of conclusions regarding this increase in the frequency of bronchogenic carcinoma. The increase cannot be due to changes in the hereditary composition of the population and must therefore be caused by changes in environmental influences. Cancer of the lung as an occupational disease would not be expected to affect appreciably the mortality figures as a whole. Pneumoconiosis has no established relationship to bronchogenic carcinoma. In this connection, careful review of available evidence on the high frequency of pulmonary cancer in the pitchblende miners of Joachimsthal and Schneeberg does not allow conclusions of causal connection to radioactivity (1 17). Exposure among workers in chromate industries, however, is related to markedly greater frequency of pulmonary cancer (131). No acceptable demonstration is available that specific infections, such as tuberculosis or influenza, have an influence on the development of pulmonary neoplasms. Among environmental influences that may play a role in the steadily increasing frequency of bronchogenic carcinoma in man, two have gained particular attention. The first are the various atmospheric contaminants from industrial smokes and fumes, exhaust fumes and dusts created by individual inhabitants, and dusts from tarred roads. Urban populations have a higher frequency of bronchogenic carcinoma than rural populations (165, 207). There is also ample laboratory evidence (31-35, 109, 110, 135) that atmospheric contaminants may contain substances that are carcinogenic when introduced into mice by injection or by inhalation. The second area of suspicion has been focused upon the smoking of tobacco. The reports of Doll and Hill (44), Doll (43a), Wynder and Graham (208), Korteweg (99), Sadowsky et al. (155), and others make it difficult, if not impossible, to deny that there is an association between prolonged, excessive smoking, particularly of cigarettes, and the occurrence of bronchogenic carcinoma, particularly of the epidermoid and undifferentiated types. The laboratory investigations as summarized by Wynder et al. (209) indicate that materials carcinogenic in rabbits and in mice can be demonstrated in some tobacco products. The present status of this important problem is well stated by one of the resolutions passed by a conference on the Endemiology of Lung Cancer (192a), held in July 1952 in Louvain under the auspices of the Council for the International Organizations of Medical Science: “The smoking of tobacco-especially cigarettes-has often been regarded as a causal factor of cancer of the lung. While it would be impossible to accept

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tobacco smoking as the only cause of cancer of the lung, there is now evidence of an association between cigarette smoking and cancer of the lung, and that this association is in general proportional to the total consumption. Further research on this subject is imperative.” The demonstration of the relationship of smoking and of other environmental factors and pulmonary cancer must be derived from direct epidemiologic studies on human populations. The follow-up studies on this problem now in progress in the United States (63a) and in England should provide the necessary definitive information. The variation in response among different species, to different chemicals, routes, and methods of administration should make it obvious that studies on man and studies on laboratory animals do not necessarily parallel. Even superficial comparison of the human bronchogenic carcinoma and the adenomatous pulmonary tumor of the mouse, rat, and guinea pig makes it evident that biologically and morphologically they are quite different. Nevertheless, such animal studies are most useful in clarifying which agents or combination of agents in the atmosphere or in tobacco are carcinogenic under laboratory conditions and therefore of possible significance in the human population. In this concept of the relationship of laboratory and clinical investigations, exact duplication of the human lesion in animals is not an essential; the skin of the rabbit, for example, may be as informative as the pulmonary tissue of strain A mice. In my opinion, however, the data already available on the epidemiology of human cancer of the lung and from the laboratory seem sufficiently impressive to urge the initiation of public education and related public health measures toward the reduction in the individual consumption of tobacco, particularly in the form of cigarettes, and toward the control of atmospheric pollution that is becoming an ever-increasing problem in many urban centers. The reader is directed to the important papers of Wynder (206), W. E. Smith (186), Hueper (go), and particularly to the reports of the Louvain conference (192a) for recent discussions and surveys of this topic, and for further bibliographic references. 2. Other Pulmonary Tumors

Two other pulmonary neoplasms of man should be mentioned in connection with the comparative pathology of human and animal tumors. The first is the bronchial adenoma, a slowly growing neoplasm that is found in approximately the same frequency in females as in males (113, 203). Bronchial adenomas show local invasion, and metastases can occur to the regional lymph nodes and even to distant organs. During the past *Added in proof. See Hammond, E. C. and Horn, D., 1954. J. Am. Med. Assoc. 166, 1316-1328.

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few years a well-founded tendency has developed to classify this growth as a low-grade carcinoma. The fact remains, however, that histologically and biologically it represents a somewhat different neoplasm from the usual bronchogenic carcinoma. The second, less frequent neoplasm or allied disease in man is pulm,onary adenomatosis, or bronchiolar carcinoma (1 13), a multicentric process manifested by cuboidal or columnar cells containing mucus lining the alveoli and projecting in papillary structures. One of the synonyms of the disease is alveolar-cell carcinoma, and the disagreement regarding the nature of the alveolar lining cell is recapitulated in discussions of this topic (60, 205). The tumor grows by extension, but metastasis to regional lymph nodes and to distant organs does occur and cannot be facilely circumvented by rediagnosing these cases. Pulmonary adenomatosis in man is morphologically indistinguishable from a disease of sheep that has been named Jagziekte, Montana Progressive Pneumonia, and Verminous Pneumonia. Its frequency has reached epizootic proportions in Iceland and in South Africa. Cowdry (39) in 1925 did not consider it a neoplasm but an “alveolar proliferation,” and at that time no metastases had been described. Bonne (28) on the other hand, referred to it as a “carcinosis caused by an infectious agent.” Dungal (47), in his extensive studies, stated that he had observed one irrefutable metastasis in a lymph node. Inoculations of filtered or unfiltered material into healthy sheep produced very few results, but the exposure of healthy sheep to the exhaled breath of sick sheep or the injection of such excretions was stated to transmit the disease readily after an incubation period of six to eight months. He concluded that the disease was probably caused by a pneumotropic virus, but the data cannot be said to have established the conclusion. Transmission to man has not been observed, and the presence of lung worms has no role in the disease in sheep. It seems reasonable to agree with Dungal’s (46) earlier statement, in that a discussion of whether Jagziekte is neoplastic or infectious in origin is rather unprofitable so long as we do not know the cause of either. The question of the relationship of pulmonary adenomatosis of man to Jagziekte of sheep, and of the resemblance of both to the pulmonary adenomatous tumor in the mouse, is as wide as the whole intriguing problem of cancer. What are the relationships between proliferative cellular processes, metaplasia, and “true” neoplasia? Are they continuous, over-lapping, or entirely different reactions of tissue? Does the demonstration of the presence of an infectious agent ips0 fact0 place an anaplastic cellular process that metastasizes into a different group of diseases? Examination of the experiences in cancer research soon reveals that no sharp division exists between certain infectious processes and neo-

TABLE I11 Comparative Fcaktrrs of Some Pulmonary Neoplasms and Related Lesions in Man and Animals Autonomy Pulmonary Lesion

Species

Bronchogenic carcinoma Bronchial adenoma Adenomatosis

Man

Pulmonary tumor Pulmonary tumor Pulmonary tumor Jagziekte

Mouse

Man Man

Rat Guinea Pig Sheep

Cell of Origin I3ronchial epithelium Bronchial epithelium Alveolar cell? Alveolar cell Alveolar cell Alveolar cell Alveolar cell

Mass Invasion

++ + ++ + + + ++

Transmission

Anaplasia

____-

Metastases Pattern

Cell

++

++

f

T

-

-

T

-

-

T

T

-

*

?

-

-

i

?

-

-

T

3

T

-

-

-

+

+ + + + +

___-

Trans- Cell-frce planta- trans- CommuniMitosis tion mission rability

++ ++ ++

8

?

?

-

P

3

?

?

-

W

T

?

7

-

-

-

+

+ +

?-

-

?-

-

+

+

31

M r

rn X

PULMONARY TUMORS IN EXPERIMENTAL ANIMALS

261

plasia, nor between so-called benign and malignant neoplasms. The conclusions maintained at present are reflections of authoritative insistence rather than of demonstrable fact. One is reminded of a statement purported to have been made by James Ewing during one of the recurrent arguments as to whether Rous sarcoma of fowl is a neoplasm or a proliferative response to an infection. His remark, in essence, was that since neoplasms were defined as new growths of unknown etiology, and since the etiologic agent in Rous sarcoma was known, Rous sarcoma by definition could not be a neoplasm! In a field that is as much a mystery as cancer is at present, the most rigid considerations must be applied to define any specific disease as belonging to the group of neoplastic diseases. Such specificity of concept immediately precludes any but the most tenuous extrapolations from one neoplastic disease to another, or conclusions of identity between morphologically similar diseases in different species of animals. At the same time, it must not remove from consideration certain related conditions and pathological processes simply because they do not fit neatly under the self-imposed criteria. One wonders, for example, why the intriguing neoplasm, for so it must be labeled on the basis of factual evidence, the transmissible lymphosarcoma of the dog (204), is no longer a subject of interest in cancer research. In Table 111 are indicated seven types of pulmonary neoplasms and allied reactions, and some of their characteristics. It is best, at this time, to consider these as separate entities, and at the same time of course to draw tentative analogies as a basis for further experimentation. Some years ago, it was proposed (173) that a “vertical” approach to the cancer problem, in which a specific neoplasm is studied from as many aspects and scientific disciplines as possible, may be more rewarding than the usual attack, in which many types of neoplasms are dealt with from the standpoint of the particular scientific discipline in which the investigator is trained. Experiences and the record of cancer research during the intervening eight years have reinforced this view. If this is more generally accepted, and long-term support of cancer research as a thirty-years’ war and not as a five-year skirmish continues, the primary adenomatous pulmonary tumors of the mouse, rat, and guinea pig would be strongly represented among the experimental neoplasms, and the bronchogenic carcinoma of man among the neoplastic diseases particularly suitable for epideminologic studies and preventive public health measures. REFERENCES 1. Andervont, H. B. 1935. Public Health Repts. (U.S.) 60, 1211-1217. 2. Andervont, H. B. 1937. Public Health Repts. ( U . S . ) 62, 212-221.

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MICHAEL B. SHIMKIN

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

hdervont, H. B. 1937. Public Health Repts. ( U . S.) 62, 304-315. hdervont, H. B. 1937. Publzc Health Repk. (Zr. S.) 62, 347-355. Andervont, H. B. 1937. Public Health Repts. ( C . S.) 62, 1582-1589. Andervont, H. B. 1938. Public Health Repts. (V.S.) 63, 229-237. Anden-ont, H. B. 1938. Public Health Repts. ( U . S.) 63, 1647-1665. Andervont, H. B. 1938. Public Health Repts. (V.S.) 63, 1665-1671. Andenont, H. B. 1939. Public Hea2th Repts. (". 8.)64, 1158-1169. Andervont, H. B. 1939. Public Health R e p k . ( U . S.) 64, 1512-1533. Andervont, H. B. 1940. J. Satl. Cancer Znst. 1, 135-145. Andervont, H. B. 1941. J . Natl. Cancer Znst. 1, 737-744. Andervont, H. B. 1945. J. 1L'atl. Cancer Znst. 6, 383-390. Andervont, H. B. 1952. Ann. N. Y. A m d . Sn'. 64, 1004-1011. Andervont, H. B., and Lorenz, E. 1937. Public Health Repts. ( U . S.) 62, 637-647. Andervont, H. B., and Lorenz, E. 1937. Public Health Repts. (C'. 8.) 62, 1931-

17. 18. 19. 20.

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49. Dunning, W. F., Curtis, M. R., and Bullock, F. D. 1936. Am. J. Cancer 28, 681-712. 50. Engel, R.W., Copeland, D. H., and Salmon, W. D. 1947. Ann. N . Y. Acad. Sci. 49, 49-67. 51. Essenberg, J. M. 1952. Science 116, 561-562. 52. Feldman, W. H. 1932. “Neoplasms of Domesticated Animals.” W. B. Saunders Company, Philadelphia. 53. Flory, C. M. 1941. Cancer Research 1, 262-276. 54. Geever, E. F., Neubuerger, K. T., and Davis, C. L. 1943. Am. J. Pathol. 19, 913-937. 54a. Glover, R. E., and Jennings, A. R. 1953. J. Pathol. Bacteriol. 66, 185-191. 55. Goldberg, S. A. 1920. J. Am. Vet. Med. Assoc. 68, 47-63. 56. Grady, H. G., and Stewart, H. L. 1940. Am. J. Pathol. 16,417-432. 57. Green, E. U. 1942. Cancer Research 2, 210-217. 58. Greenstein, J. P. 1954. “Biochemistry of Cancer,” 2nd ed. Academic Press, New York. 59. Greenstein, J. P., and Shimkin, M. B. 1945. Research Conference on Cancer. Publ. Am. Assoc. Advance. Sci., p. 222. 60. Griffith, E. R., McDonald, J. R., and Clagett, 0. T. 1950. J. Thoracic Surg. 20, 949-960. 61. Gross, L.,Gluckman, E. C., Kershaw, B. B., and Posselt, A. E. 1953. Cancer 6 , 1241-1243. 62. Guyer, M. F., and CIaus, P. E. 1947. Cancer Research 7 , 342-345. 63. Haaland, M. 1911. Fourth Sci. Rept. Invest. Imp. Cancer Research Fund, pp. 1-114. 63a. Hammond, E. C. 1954. Connecticut State Med. J . 18,3-9. 64. Harding, H. E., and Green, H. N. 1946-47. Annual Report, Yorkshire Council British Empire Cancer Campaign, p. 8. 65. Heidelberger, C., and Jones, H. B. 1948. Cancer 1, 252-260. 66. Heidelberger, C., and Weiss, S. M. 1951. Cancer Research 11, 885-891. 67. Henshaw, P. S., and Meyer, H. L. 1944. J. Natl. Cancer Znst. 4, 523-525. 68. Henshaw, P. S., and Meyer, H. L. 1945. J. Natl. Cancer Znst. 6, 415-417. 69. Heston, W. E. 1940. J. Natl. Cancer Znst. 1, 105-111. 70. Heston, W. E. 1941. J . Natl. Cancer Inst. 2 , 127-132. 71. Heston, W. E. 1942. J. Natl. Cancer Inst. 3, 69-82. 72. Heston, W. E. 1942. J. Natl. Cancer Inst. 3, 303-308. 73. Heston, W. E. 1948. Advances i n Genetics 2, 99-125. 74. Heston, W. E. 1949. J. Natl. Cancer Znst. 10, 125-130. 75. Heaton, W. E. 1951. Growth 10,23-46. 76. Heston, W. E. 1952. Am. J. HumanGenet. 4, 314-331. 77. Heston, W. E. 1953. Proc. SOC.Exptl. Biol. Med. 82,457-460. 78. Heston, W. E., and Deringer, M. K. 1947. J. Natl. Cancer Znst. 7 , 463-465. 79. Heston, W. E., and Deringer, M. K. 1949. J. Natl. Cancer Znst. 10, 119-124. 80. Heston, W. E., and Deringer, M. K. 1951. J. Natl. Cancer Inst. 12, 361-367, 1951. 81. Heston, W. E., and Deringer, M. K. 1952. J. Natl. Cancer Inst. 13,705-718. 82. Heston, W. E., Deringer, M. K., Hughes, I. R., and Cornfield, J. 1952. J . Natl. Cancer Znst. 12, 1141-1157. 83. Heston, W. E., and Dunn, T. B. 1951. J . Natl. Cancer Inst. 11, 1057-1171. 84. Heston, W. E., and Lorenz, E. 1953. Cancer Research 13, 573-577.

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