observations on animals painted with tobacco tar · observations on animals painted with tobacco...

OBSERVATIONS ON ANIMALS PAINTED WITH TOBACCO TAR

KANEMATSU SUGIURA, D.M.Sc.

(From the Huntington Fund for Cancer Research, Memorial Hospital, New Y o r k )

The importance, in the etiology of cancer, of various agents, such as roentgen rays (1, 2), radioactive substances (3, 4), ultraviolet light (S), certain animal parasites (6, 7), coal tar (8 ) , and some hydrocarbons (9, 10, 11) which produce prolonged or chronic irritation is now generally recognized. Such studies have not been limited to experimental animals, and a number of writers have suggested a relationship between these agents and certain forms of occupational cancer. The high incidence of cancer of the lip, tongue, and throat in men as compared to the infrequency of such cancer in women has frequently been attributed to persistent irritation of the epithelial surfaces by tobacco tar, to which men have been exposed to a much greater degree.

Stimulated by these observations, several investigators have studied the action of tobacco tar and smoke in different species of animals. In 1911, Wacker and Schmincke 12) succeeded in producing an atypical proliferation in rabbits' ears after application of tobacco tar, obtained from pipes, mixed with crude paraffin oil. Helwig (13) found that the ethereal extracts of tobacco tar obtained from the bowls of briar pipes and combustion products of tobacco distilled over at temperatures between 400 and 500" C. did not contain any carcinogenic substance capable of producing unlimited growth of epi- thelium in mice. Tobacco tar when mixed with olive oil and injected into the ears of rabbits caused atypical epithelial proliferation, but in no instance did it produce actual malignant transformation. Chikamatsu ( 14) painted the skin of mice and the ears of rabbits with tobacco tar and found it to be inactive on mice though it caused the development of a cancroid on a rabbit's ear 225 days after the beginning of treatment.

Recently Roffo (15) carried out extensive experiments on rabbits, using three distilled products of tobacco : watery distillate at 100-120" C., thick Iiquid at 120-350" C., and the residue. These products were applied daily to the ears of 60 rabbits for ten months, 20 animals being used for each prod- uct. None of the rabbits treated with the watery distillate developed tumors, but 95 per cent of those treated with the thick liquid and 70 per cent of those with the residue had squamous epitheliomas. The carcinogenic agent present in the tobacco tar is said to be spectroscopically identical with carcinogenic hydrocarbons related to benzanthracene ( 16). These recent experiments afford a confirmation of Roffo's earlier reports (17) on the production of malignant growths in rabbits and rats by tobacco tar.

Since the carcinogenic substance of coal tar is found largely in the fractions with a high boiling point (above 550 ' C.), Cooper, Lamb, Sanders and Hirst (18) determined the average temperature of tobacco burning in pipes. Under conditions of normal smoking the temperature of combustion was found to vary from about 370 to 590 ' C. according to the variety of tobacco, but

41

42 KANEMATSU SUGIURA

with vigorous smoking the temperature reached 700" C. Employing a spe- cially constructed apparatus, they prepared crude tar products at temperatures between 400 and 800" C. Since these tar products were extremely toxic to mice, their chloroform extracts were treated with normal hydrochloric acid and sodium hydroxide solutions, The purified tar products were applied to the skin of the mice at intervals of about three days in alcohol, benzene, or glycerine solution. Among 50 mice thus treated, only one developed an epithelioma, sixteen months later, and this with a tar produced at 400-500" C.

Lu-Fu-hua (19) obtained a thick tar by collecting the distillate from the combustion of tobacco at a temperature of 400-500" C. in a series of bottles containing water. He applied this tar to the right ears of four rabbits and at the same time painted the left ears with coal tar two or three times weekly. All four rabbits developed carcinoma on the left ears, and three of them carcinoma on the right ears as well, after 148 to 182 days. When large amounts of cholesterol were introduced into rabbits which had been subjected to prolonged painting with tobacco tar, the appearance of cancer was hastened, but cholesterol treatment before tar painting had no accelerating influence upon the development of cancer.

At about the same time, Schurch and Winterstein (20) re.ported their failure to produce cancer in mice and rabbits by painting the skin or the mucous membrane with nicotine-free tobacco tar, alone or in combination with mechanical or thermal injury. In rabbits, however, that had suffered con- stitutional damage from a diet rich in cholesterol (egg yolk) and coal tar painting, tobacco tar warts and carcinoma could be produced. Preliminary treatment consisted in the administration of a cholesterol-rich diet and weekly painting of one ear with coal tar. After several months papillomas appeared and the coal tar painting was discontinued. Tobacco tar was then applied twice weekly to the non-treated ear. Among 7 survivors out of 10 rabbits, 5 developed papillomas after 198 to 700 days, and one developed carcinoma after 344 days. In a subsequent paper (21) the authors reported the occur- rence, after four years of tobacco tar application in a similar manner, of a squamous carcinoma on the ear, a papilloma on the nictitating membrane, niultiple hemangiomata of the intestine, and a spindle-cell sarcoma of the rectum in one rabbit. No metastases are recorded.

Kinoshita ( 2 2 ) reported the work of Taki, who collected tobacco tar from Japanese pipes and dissolved it with ether. The ether extract was treated with hydrochloric acid and repeatedly washed with water. The residue, after evaporation of ether, was applied twice weekly to the skin of mice. Of the 104 mice, 10 survived from 110 to 130 days, 2 developed squamous epitheliomas and 3 others papillomas. Campbell (23) observed that prolonged exposure of mice to fumes from cigarettes (seven hours daily on five days each week) increased the incidence of primary lung tumors but this increase he attributed to the longevity of the animals, which survived much longer than the controls. Painting the skin of mice with the tarry mass obtained by combustion of cigarettes failed to produce any cancerous change, although one mouse developed a suspicious wart-like growth.

The use of differently distilled products of various kinds of tobacco and

OBSERVATIONS ON TOBACCO TAR 43

of animals of different strains have led to varying results. In view of these facts it seems desirable to give an account of our own experiments in which we produced a single malignant growth in an animal by long continued ex- ternal application of tobacco tar.

PREPARATION OF TOBACCO TAR

A mixture of American and Turkish leaf tobacco was placed in an iron retort (Fig. 1) and heated for four hours with a Meker burner. The distillate was collected at a temperature of 100-500 ' c. The residue was then heated with a blast burner for four hours and the distillate collected between 500 and 900 C.' The combustion of 110 grams of finely cut tobacco yielded

FIG. 1 . IRON RETORT FOR COMIKSTION DISTILLATION OF TOBACCO

about 27 C.C. of the first distillate and upon heating with the blast burner gave about 7 C.C. additional distillate. Of the first distillate, about 23 C.C.

consisted of a yellowish brown translucent liquid (nicotine fraction), and about 4 C.C. of a very dark brown, almost black, oily liquid (oil, fat, resins fraction). The second distillate was a thick, very dark brown, almost black, oily liquid (resins fraction).

The first distillate was separated into two parts, the watery liquid and the oily liquid, and their carcinogenic activity was tested separately.

All three products gave a strong alkaline reaction and had a disagreeable odor. They were also extremely toxic. Subcutaneous or intramuscular in- jection of 0.1 C.C. of the tobacco distillates killed a young adult rat or mouse within one hour. The animals died with convulsions.

1 The approximate temperatures were determined by inserting a thermometer in sea sand contained in the iron retort and by the color produced on the iron retort by heating.


EXPERIMENTAL In the first experiment, started on Feb. 3, 1937, 0.02 gm. of the tobacco

distillates were applied twice weekly to the skin of the interscapular region of mice. A small area of skin was prepared by shaving and by subsequent trimming of the hair when necessary. The inside surface of the ears of rats and rabbits was treated three times weekly with 0.05 gm. and 0.2 gm. of the distillates respectively.

For this experiment we used 186 Bagg albino mice, in which the incidence of spontaneous mammary tumors in breeding females is about 18 per cent; 30 dba mice, in which the incidence of spontaneous mammary tumors in breeding females is about 65 per cent; 30 C57 black mice, in which the incidence of spontaneous mammary tumors in breeding females is less than 1 per cent; 35 albino rats of Wistar Institute stock, in which the incidence of spontaneous mammary tumors in breeding females is less than 1 per cent, and 2 2 common stock rabbits, of both sexes. The mice and rats were two to three months old at the time of the first application of tobacco tar. All female mice and rats were virgins.

Rats and mice received a common diet of Purina dog chow and lettuce, and rabbits were given Platt's complete rabbit pellets and lettuce. Water was allowed in unlimited amount.

The tar painting was continued even after development of a wart-like growth at the site of the paintings. When an animal died it was carefully autopsied and new growths, as well as suspicious material, were sectioned for histologic examination.

Experiments with Bagg Albino Micc: In this series of experiments 186 inice were used, of which 168 lived over 90 days, or beyond the minimum period of time required to elicit a neoplasm by painting with coal tar. Of this number, 148 mice lived 150 days, 115 lived 200 days, 78 lived 250 days, 48 lived 300 days, 32 lived 350 days, 2 2 lived 400 days, and 20 lived 500 days or over. Of the 168 mice that lived over 90 days, 44 were painted with the watery liquid of the first distillate, 57 were painted with the oily liquid of the first distillate, and 67 were painted with the second distillate.

The results show that of the numerous mice painted with tobacco tar, only one developed a tumor at the site of application (Fig. 2), and this with the second distillate collected at 500-900" C. In this female mouse, a wart-like growth was discovered on the 315th day. Despite continued tar painting, it grew very slowly. The animal died thirty-five days after the tumor was first observed. Post-mortem examination revealed no metastasis. Histologic examination showed an epithelial papilloma with considerable keratinization of the superficial layer. There was no evidence of ulceration. The basal layer of the epidermis was intact and showed no tendency to infiltrate the under- lying connective tissue. In one area there was loosening of the epithelial cells in the basal portion with atypical growth and numerous mitoses, indicating squamous carcinoma (Fig. 3).

For the sake of comparison, a number of Bagg albino mice of nearly the same age were similarly painted with coal tar (National Aniline and Chemical Co., New York). The results obtained were quite opposite to those obtained

F I G . 2. SKIN TUMOR I N A MOUSE AT T H E SITE OF REPEATED TOBACCO TAR PAINTINGS

FIG. 3. SECTION OF THE TUMOR (FIG. 2 ) SHOWING LOOSENING OF TIIE EPITHELIAL CELLS 19;CIp BASAL PORTION WITH ATYPICAL GROWTH AND NUMEROUS MITOSES INDICATING

SQUAMOUS CARCINOMA 45


with tobacco tar. Of 57 mice, 48 lived from 75 to 497 days, and of this number 36 ( 7 5 per cent) developed progressively growing tumors at the site of tar painting. The earliest time of appearance of a papilloma was 75 days and the latest was 309 days. Fifty per cent of the papillomas appeared between the 149th and 200th day after the initial painting. In 31, or 86 per cent of the 36 papillomatous growths, squamous carcinoma had developed, This was verified by histologic examination of the tumors.

In the course of this investigation, determinations were made on the influence of cholesterol upon the production of skin tumors. This was done by maintaining a number of mice on a diet’ containing 10 per cent of pow- dered beef brain or egg yolk during the entire period of tobacco tar painting. Forty Bagg albino mice were used, of which 28 lived from 90 to 180 days. This cholesterol-rich diet had no accelerating influence on the development of tobacco tar cancer.

Experiments with dba Mice and C57 Black Mice: A number of investigators (24, 25) have clearly shown that there is marked difference among various strains of mice in susceptibility to the carcinogenic action of polycyclic aromatic hydrocarbons. For this reason experiments duplicating those with the Bagg albino mice were conducted with dba mice and C57 black mice. The animals were painted twice weekly with the mixture of the oily liquids of the first and the second distillates. Unfortunately the dba mice did not tolerate the tobacco tar paintings and died early in the course of the experiment without showing any significant change on the painted area (none of the 30 mice lived over 31 days). Thirty C57 black mice tolerated repeated paintings of tobacco tar quite well. The animals lived from 80 to 275 days, 21 surviving from 163 to 275 days. No tumors were found in any mice.

Experiments with Rats and Rabbits: Although rat skin is extremely re- sistant to the carcinogenic action of coal tar and hydrocarbons, the ears of rats are highly susceptible to the cancer-producing effect of ultraviolet rays (26, 27).

A series of experiments was conducted to determine whether the ears of rats share the exemption from the carcinogenic action of tobacco tar that is shown by the skin of mice. The internal surface of the ears was painted with the combined dark brown oily liquids of the first and second distillates, dis- tilling at 300-900’ c., two to three times weekly for 48 to 65 weeks. Thirty of 35 rats survived the long-continued tar paintings but showed no evidence of tumors.

Experiments duplicating those with rats were made with rabbits. Tobacco tar was applied to the inside of the ears of 2 2 rabbits three times weekly, a much greater amount of tar being used at each painting (0.2 gm.) than for the other animals. After fifty-two to ninety-five weeks, repeated tobacco tar paintings had caused no atypical epithelial proliferation.

EBect of Tobacco Tar and Nicotine on Viability of Mouse Sarcoma and Rat Carcinoma: In addition to nicotine, other irritating factors, in the form At,ddehydes, ketones, ammonia, furfural and pyridine, are present in the &-!illed tobacco oil. To determine whether these substances are capable of inhibitjqg.t-r growth, the following experiments were carried out. .... ..

,&?@cJ@r.&aE8O per cent, whole milk powder 19 per cent, irradiated yeast 1 pr cent.

OBSERVATIONS O N TOBACCO TAR 47

Fragments of fresh mouse sarcoma 180 and Flexner-Jobling rat carcinoma were placed in 2.0 C.C. portions of our tobacco distillates. These media had neither been buffered to pH 7.4, nor been made isotonic. The weighing bottles containing the tumor fragments were kept in a refrigerator at 4-5" C. for six to twenty-four hours. At the end of these periods, the tumor fragments were removed, washed twice in Locke-Ringer solution, and then inoculated into normal mice or rats.

The growth capacity of the mouse sarcoma and of the rat carcinoma was completely destroyed when tumor fragments were immersed in tobacco tar for six hours or more at 4-5" C.

Similar experiments were made with pure nicotine solution (Eimer and Amend). Here also immersion of fragments of fresh mouse sarcoma 180 and Flexner-Jobling rat carcinoma in nicotine solution for one, six, and twenty- four hours at 4-5 ' C. produced complete inhibition of growth. This study consisted of 5 groups of experiments involving a total of 80 mouse sarcoma implants and 50 rat carcinoma implants.

Our studies on the influence of hydrogen ion concentration upon the viability of transplantable tumors indicated that the growth capacity of mouse sarcoma 180 is completely destroyed by immersion in a Locke-Ringer solution at pH 4.0 or 10.0 for twenty-four hours at a temperature of 4-5' C. (28), while the viability of the Flexner-Jobling rat carcinoma is completely destroyed when the pH of the medium is 5.0 or 9.0 (29). Both pure nicotine solution and our tobacco tar are strongly alkaline. Since the reaction of the media is a possible factor in the death of tumor tissues, the nicotine solution was adjusted to approximately pH 7.4 by adding 2.2 C.C. of 0.1 N HCI for each 5.0 C.C. of a 1.0 per cent solution. As before, fragments of fresh tumor tissue were placed in 2.0 C.C. portions of the neutral 1.0 per cent nicotine solution. After standing for twenty-four hours at 4-5" C., the tumor fragments were implanted into normal mice or rats in the usual way. Four groups of experiments were conducted, involving a total of 40 mouse sarcoma implants and 40 rat carcinoma implants. The neutral nicotine solution appeared to have no inhibitory effect on the growth capacity of mouse sarcoma 180 and Flexner-Jobling rat carcinoma. The number of takes and the rate of tumor growth after treatment with the nicotine solution were practically the same as for untreated tumor grafts.

The preceding experiments were repeated with tobacco tar. The tobacco distillates were buffered to approximately pH 7.4, by the addition of proper amounts of 0.2 M KH,PO, and 0.2 M KOH. Small pieces of tumor tissue were placed in these solutions and left for twelve to twenty-four hours at 4-5" C., before inoculation. The results obtained from these experiments were not clear cut, due partly to difficulty in obtaining a proper pH of the medium by the colorimetric method. However, the inhibitory effect of the buffered tobacco distillates on the proliferating capacity of mouse sarcoma 180 and Flexner-Jobling rat carcinoma was much less than that of distillates which were not buffered.

The results of these comparative experiments show that the inhibitory action of our tobacco distillates and pure nicotine solution on the proliferating

48 KANEMATSU SUGlURA

capacity of mouse sarcoma 180 and Flexner-Jobling rat carcinoma is due not to the nicotine, but is attributable solely to the alkalinity of the media.

DISCUSSION In comparing the results obtained from experiments with tobacco tar and

coal tar it will be noted that the carcinogenic activity of tobacco tar is very much less than that of coal tar. Only 1 (0.6 per cent) of 168 mice which had been subjected to repeated painting with tobacco tar for from 90 to 500 days developed a squamous carcinoma at the site of application. On the other hand, 36 out of 48 mice painted with coal tar developed papillomas on the skin within 75 to 309 days. Thirty-one of these 36 mice, or 65 per cent of the total number of animals, developed squamous carcinoma. Furthermore, the average induction time in the coal-tar-treated animals was very much less than for those treated with tobacco tar. Since the amount of tar used at each painting was approximately the same, namely 0.02 gm. for the two groups, the difference is not attributable to the dosage factor.

Our results with rabbits are not in conformity with the findings of Roffo ( 15) , who succeeded in producing a large number of papillomas and true malignant growths after continued application of tobacco tar. Since Roffo's results seem undoubtedly significant, it is possible that some other factor than tobacco is responsible for the discrepancy. Roffo subjected tobacco of various kinds to fractional combustion distillation in a stainless steel retort, whereas we used an iron retort. I t is difficult to believe that any hydrocarbons with a phenanthrene nucleus, which Roffo claims are contained in his combustion products of tobacco, should break up by our method of distillation. In addition, Kennaway (30) has shown that the carcinogenic substance of coal tar is not found in any appreciable quantity below 550" C.

Little information is available concerning the presence in tobacco tar of carcinogenic hydrocarbons identical with those contained in coal tar. In 1932, Cooper, Lamb, Sanders and Hirst (18) subjected tobacco tar products to spectrographic analysis to disclose a possible carcinogenic agent but reported in- conclusive results. I t was said, however, that all the samples of tar, whether obtained at low or high temperature, exhibited fluorescence. Schiirch and Winterstein (20), in 1935, reported that the fractions obtained from tobacco tar did not contain polycyclic aromatic hydrocarbons related to the carcinogenic hydrocarbons contained in coal tar. Recently Roffo (15, 16) made in- tensive spectrographic examinations of products of combustion of tobacco and found hydrocarbons with a phenanthrene nucleus identical with that in the carcinogenic hydrocarbons present in coal tar. Roffo, however, made all his spectrographic analyses with impure complex mixtures. No definite conclu- sions can be drawn from the absorption bands of these bodies until the specific hydrocarbon is isolated in pure chemical form.

Another factor which might account for the disagreement between our findings and those of Roffo (15) and Taki ( 2 2 ) is the use of animals of different strains. I t is now known that animals of various strains possess marked differences in susceptibility to the carcinogenic action of hydrocarbons.

From the fact that only one squamous carcinoma has been obtained in the

OBSERVATIONS O N TOBACCO TAR 49

course of subjecting a large number of mice to the action of tobacco tar, compared with the very high incidence of cancer in mice treated with coal tar, it seems reasonable to classify tobacco tar as a weak carcinogenic agent.

SUMMARY 1. Prolonged painting of the skin of mice with combustion products of

tobacco distilled over at temperatures between 100 and 900’ C. produced a single instance of squamous carcinoma among 168 Bagg albino mice that lived from 90 to 500 days.

2. Painting the skin of C57 black mice and dba mice, as well as the ears of rats of Wistar Institute stock and of rabbits of common stock, with tobacco tar failed to produce any cancerous change.

3. The proliferating capacity of mouse sarcoma 180 and the Flexner- Jobling rat carcinoma was unaffected by tobacco tar or pure nicotine solution when the solutions were adjusted to pH 7.4.

1 . 2.

3. 4. 5. 6. 7 . 8.

9. 10. 1 1 .

12,

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