ON THE MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS

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1 ON THE MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS IZRAEL RIEGER (From the Research Institute of the Royal Cancer H osjjital (Free), London, S. W. 3) INTRODUCTION The object of the experiments described here was to investigate the fate of carcinogenic hydrocarbons when they are acted upon by tissues, and also to seek for any relation between normal tissue, hydrocarbon, and neoplasm, which might help to explain cancer production. At presentt it is not known whether a carcinogenic hydrocarbon acts as such or whether it is first converted by the tissues into an active derivative. By analogy with certain drugs, prolonged stability in the body does not necessarily imply pharmacological inactivity, but does indicate the length of time over which the drug can act. Some carcinogenic hydrocarbons are remarkably stable to ordinary laboratory reagents; for example, 1: 2: 5:6-dibenzanthracene can be purified by washing with concentrated H"S04 without loss of potency. The mechanisms in the body which deal with foreign substances might render the hydrocarbon soluble in preparation for elimination, or transport it to the liver for detoxication, or deposit it in some slowly metabolising tissue such as bone. But tumours caused by hydrocarbons arise at the site of application, if we except the lung tumours occurring in mice to which these compounds have been applied. These findings suggest that carcinogenesis requires the activity of hydrocarbons per se and not of their derivatives, otherwise one would expect tumours to arise somewhere in the excretory organs. Further evidence favourable to this view will be found in the results recorded below. EXPERIMENT I: THE MINIMUM DOSAGE FOR TUMOUR PRODUCTION During the course of experiments on the persistence of hydrocarbons, tumours of the skin were observed in mice painted biweekly with 1: 2:5:o-dibenzanthracene for only sixteen weeks, but not in others painted thus for eight weeks only. This difference may possibly be a mere quantitative effect, i.e. the amount of 1: 2: 5: 6-dibenzanthracene applied in eight weeks of biweekly painting may be below the threshold amount. Were this explanation correct, an increase in the dilution of a carcinogenic solution could be compensated by a corresponding increase in the frequency of application. In Table I are given the details of a test made with 0.9 per cent solutions of agent applied once a week and of 0.3 per cent solutions of the same substance applied thrice a week. Solutions A and C were 0.3 per cent, and solutions B and D 0.9 per cent, dilutions in benzene of crystalline fractions obtained from 522

2 MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS 523 pitch consisting chiefly of 1:2-benzpyrene; the pairs of solutions A and B, and C and D, were prepared from two different fractions. The yield of tumours, their date of appearance (T), and the survival rate of the mice are shown in the table. TABLE I: Experiment I. The Minimum Dosagefor Tumour Production Solution A Solution B Solution C Solution D 0.3 per cent 0.9 per cent 0.3 per cent 0.9 per cent Thrice weekly Once weekly Thrice weekly Once weekly Day of experiment Appear- Appear- Appear- Mice ance Mice ance Mice ance Mice Appearance alive of alive of alive of alive of tumours tumours tumours tumours ---- II T (159) T (137) T (167) T (193) 9 T (193) 9 T (193) 9 T (217) T (217) TTT(217) T (217) T (245) T (245) T (273) T (291) T (291) T (291) (also 2 tumours T (336) T (336) date uncertain) I T (352) I Average day of appearance of tumour T = appearance of tumour on day indicated in parentheses. The very similar average latent periods (237, 240, 230, 236 days) of tumour production in these series are noteworthy. The high death rate with A in the early part of the experiment and the comparatively low death rate with D probably account for the difference in the total numbers of tumours in these two series. The conclusion appears to be that a threefold variation in the concentration-frequency relation has no appreciable effect upon the carcinogenic response. The limits within which this relation operates have not yet been worked out for painting experiments. Thus in the experiment referred to above, where tumours were produced in mice by painting for sixteen weeks (but not in those painted for only eight weeks) with 1:2:5 :6-dibenzanthracene, the amount of hydrocarbon required for tumour production was of the order of 1 to 2 mg. In this laboratory we have obtained tumours of the skin in

3 524 IZRAEL HIEGER mice which were painted for two years with per cent solution of 1: 2: 5: 6-dibenzanthracene (1); there the total dosage must have been of the order of 0.1 mg. of hydrocarbon per mouse.' Hence it seems that, over much longer periods, the duration of treatment is a decisive factor in tumour production rather than quantity of hydrocarbon alone. PERSISTENCE OF CARCINOGENIC AND NON-CARCINOGENIC HYDROCARBONS IN THE ANIMAL BODY Experiment II: Subcutaneous Injection in Rats of a Solution in Lard Chalmers (3), investigating the fate of 1: 2: 5: 6-dibenzanthracene in chickens, found that less than 0.1 mg. was left fourteen days after an injection of 1 mg. in solution in egg yolk fat. Apparently 0.1 mg. was near the limit of sensitiveness of his method. Berenblum and Kendal (4), using injections of colloidal 1: 2: 5:6-dibenzanthracene into the pectoral muscle of chickens, found that after eighteen months less than 0.02 mg. was left from 0.6 mg. injected. Using mice, they found that, if 2 mg. of 1: 2: 5: 6-dibenzanthrecene were injected into the peritoneum, some of the hydrocarbon could stili be detected after 160 days. If the 1: 2: 5: 6-dibenzanthracene were used as a solution in lard, all but 0.02 mg. was destroyed in sixty days, but if the hydrocarbon was injected in colloidal suspension, then 0.5 mg. remained after 160 days. Both Chalmers and Berenblum measured quantitatively the gradual decrease in the amount of 1: 2: 5: 6-dibenzanthracene, but the inclusion of parallel tests with non-carcinogenic hydrocarbons would have greatly increased the value of their experiments. The discovery of some difference in the capacity of the body to deal with these two types of compounds might well throw light on the carcinogenic process. When applied in benzene solution to the back of a mouse from a small brush, 1: 2: 5: 6-dibenzanthracene spreads rapidly, no doubt dissolving in the skin fat and forming a thin film which must be in a very suitable condition for action by tissue enzymes. When, however, the hydrocarbon is injected subcutaneously, it is usually employed in solution in a fat, which is itself not always rapidly removed from the site of injection. In fact, it is quite possible that the detection of say 1: 2: 5: 6-dibenzanthracene in tissue after the hydrocarbon has been injected in solution in a fat is an estimate of the resistance to removal of the comparatively large amount of solvent rather than an estimate of the stability of the hydrocarbon. In one experiment, for example, a lard solution of 1:2-benzanthracene was seen to spurt from the subcutaneous tissue of a rat on dissection three months after a single injection of 6 mg. of the hydrocarbon dissolved in 2 c.c. lard. In all the experiments described below on persistence of hydrocarbons in the tissues, 1: 2-benzanthracene was used as a control upon 1: 2: 5: 6-dihenzanthracene. Since the chemical behaviour of these two hydrocarbons is very similar, the hope was entertained that any single detectable difference in be- 1 That very minute quantities of a hydrocarbon may be carcinogenic is shown by the remarkable results obtained by Shear (2), who found that a sarcoma could be produced in a mouse by less than mg. of 1: 2: 5:6-dibenzanthracene.

4 MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS 525 haviour in vivo might give a clue to their remarkable difference in carcinogenic potency. The results shown in Table II give the impression that 1: 2: 5: 6-dibenzanthracene is more resistant to elimination or alteration in the animal body than is 1:2-benzanthracene. TABLE II: Experiment I I. Persistence of Hydrocarbons isubcutaneous Injection in Rats) Mg. hydrocarbon injected, in 2 c.c, lard Weeks after injection I I 2 I 3 I 4 I s I 61 7 I [II U [ S [ f IIA.. Mixed Stock Rats O.OS II _~_I_J_J J_J_I_I_J_J_J 1 JJ DB B.: J DB - Il ~---TI DB + II I = Oil II -\T\-+-\-' I ~I (in.1 c.c, lard) RII = /B: Wislar Rats IIC: M'ixed Stock Rals 6.0 DB II 23rd week + ± + 30th week + R = 1 : 2-henzanthracene. DB = 1 : 2 : 5 : 6-dibenzanthracene. Total numbers of rats examined: B 26, DR 28. Table II shows the results of tests in 54 rats for the persistence of these two hydrocarbons in the tissues; of these animals, 26 had received 1:2-benzanthracene and 28 1: 2: 5: 6-dibenzanthracene. In all the six pairs of tests in which the behaviour of the two compounds differs, it is the 1: 2-benzanthracene which is either absent (5 cases) or doubtful (1 case), while the 1:2: 5: 6-compound is still present in all six cases. Experiment II C requires some further consideration on this point. 1: 2 benzanthracene is absent in three stages and present only in traces in a fourth, whereas 1: 2:5:6-dibenzanthracene is still prominent in all four comparable animals. Nevertheless, the irregular disappearance and reappearance of the 1: 2-compound in the series of observations suggests that the fate of an injected hydrocarbon depends largely upon the dispersal of fatty solution in the subcutaneous tissue. A mass of injected fat will presumably persist much longer when exposing little surface for attack, and the exact disposition of such material is difficult to control when making the injection.

5 526 IZRAEL HIEGER Technique: Rats were given single injections (2 c.c.) of the solutions subcutaneously, and the position of the injection was then marked with picric acid solution. The rats were kept under ordinary laboratory conditions until they were killed (ether and bleeding) at the time indicated in the table. The skin around and including the marked area was removed along with the subcutaneous tissue and the peritoneal wall, with carefully cleaned instruments. In these experiments the instruments and vessels were tested for cleanliness by first boiling them for a few minutes in c.c. of non-fluorescent benzene which is prepared by doubly fractionating ordinary laboratory benzene. The articles were considered clean if, after boiling and concentrating this benzene to 1-2 c.c., no appreciable fluorescence was observed when it was transferred to a 2" X lit" tube held in filtered ultraviolet light. The tissue was minced thoroughly and dried in vacuo for at least a week, extracted by refluxing with non-fluorescent benzene for three hours, and the extract concentrated to 1-2 c.c, and tested for hydrocarbon by photographing the fluorescence spectrum. The fluorescence spectrum has some advantages over the ultraviolet absorption method: it gives quicker results, which are far simpler to interpret and, moreover, in the presence of other fluorescing substances the fluorescence spectrum of 1:2-benzanthracene, at any rate, is only slightly affected, whereas in the case of absorption spectra admixed substances may seriously alter the spectrum of the hydrocarbon in question (Hieger, 1936). The difference in behaviour between 1:2-benzanthracene and 1:2:5 :6-dibenzanthracene, shown in Table II, is actually greater than appears from single inspection of the figures there given, because the former compound is more fluorescent than the latter. A series of tests was made with a benzene extract of dried rat tissue to which the hydrocarbons were added. The limit of detectability was in the region of 1 part of hydrocarbon in from 2 to 3 million parts of diluent; the bands of 1:2-benzanthracene were still visible at a dilution where the bands of 1: 2: 5: 6-dibenzanthracene had almost disappeared into the general background of deposit on the photographic plate. Unless otherwise indicated, the results of the tests are given as positive or negative without quantitative implication (Table II). In Experiment II two of the rats which had been injected with 1: 2:5:6-dibenzanthracene developed sarcomata. Experiment Ill: Application to the Skin of Mice of a Solution in Benzene Technique: Mice were painted biweekly on the interscapular region of the skin with 0.3 per cent solution of the hydrocarbon just as in a test for carcinogenic action, except that the treatment was stopped in the different series after the period stated in the table (Table III). At weekly intervals after the last painting, mice from the different series were killed (ether and bleeding) and a large part of the skin of the back, including the painted area, was removed, minced, dried for at least a week in vacuo, and then extracted and tested as in Experiment II (Table II). The results of the tests are shown in Table III. In none of the series is either hydrocarbon detectable four weeks after the last application to the skin, and no difference between the two compounds in rate of disappearance was detected.

6 MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS 527 In the series painted with 1: 2: 5: 6-dibenzanthracene for sixteen weeks, 2 mice developed papillomata twenty-two weeks after the last. painting, i.e. eighteen weeks after the four-week period (see Table III) in which 1: 2:5: 6 dibenzanthracene had ceased to be detectable in other mice of the same series. Well advanced tumours were observed in the thirty-two week series, two weeks before the last painting, that is about eight weeks before the tumour appeared in the sixteen week series. T ABLE III: Experiment I II. Persistence of Hydrocarbons (A pplication to Skin of Mice) Weeks after last painting Painted for 1 week DB B ~- Painted for 2 weeks DB ± B ~-~ Painted for 4 weeks DB - B Painted for 8 weeks DB - B "--- Painted for 16 weeks DB - B Painted for 32 weeks DB - B - B = 1 : 2-ben7.anthracene. DB = 1 : 2 : 5 : 6-dibenzanthracene. It is curious that the time required for disappearance of the hydrocarbon should not be affected by the number of applications nor by the length of time during which the skin has been treated. Moreover, the results in the table show that both the strongly carcinogenic and the practically non-carcinogenic hydrocarbon behave in the same way, and hence carcinogenic action can hardly depend upon the rapid chemical interaction and destruction of the hydrocarbon. The rapid disappearance of the hydrocarbons is probably not a simple surface oxidation since 1:2: 5: 6-dibenzanthracene, when spread in thin films on filter paper exposed to air in an empty mouse cage, is not destroyed for six and a half weeks at the very least. The polycyclic aromatic hydrocarbons are slowly oxidised by prolonged exposure to air but far more rapidly if the reaction is catalysed by ultraviolet light (Boyland, 6). EXPERIMENT IV: ARE CARCINOGENIC AGENTS MUTUALLY REPLACEABLE? While sarcomata can be evoked by hydrocarbons as different structurally as chrysene and 1:2: 5: 6-dibenzanthracene, the skin of the mouse can, on the other hand, differentiate between two methyl derivatives of 1: 2-benzanthracene where the difference is but a single unit of position on the ring. That the

7 528 IZRAEL HIEGER carcinogenic activity and chemical structure should in some groups of compounds be so specifically related, yet in another sense be so unspecific, is at present unexplained. This non-specificity suggested that two or even more carcinogenic agents might be replaceable. In Experiment I tumours arose in mice painted biweekly with 1: 2: 5: 6 dibenzanthracene (0.3 per cent solution in benzene) for sixteen weeks, but not in mice painted for eight weeks. In Experiment IV three series of 10 mice each were set up. A. Painted biweekly for eight weeks with 1:2-benzpyrene. B. Painted biweekly for eight weeks with 1: 2: 5: 6-dibenzanthracene. C. Painted biweekly for eight weeks with 1: 2-benzpyrene, then for eight weeks with 1: 2:5 :6-dibenzanthracene. (Both hydrocarbons were dissolved in benzene at 0.3 per cent concentration.) A very small papilloma appeared in one mouse five weeks after the last painting in series C, and in a second mouse in the seventh week. By the tenth week these papillomata had developed into two large tumours 1 em. in diameter and were found on examination to be epitheliomata. In series A, two minute warts appeared which were not visible eleven days after they were first observed. No other tumours developed in the A or B series. Hence in series C a summation of the effects of the two compounds appears to have taken place. SUMMARY 1. Four series of mice were painted with benzene solutions of 1: 2-benzpyrene. In two series 0.3 per cent solution was applied three times a week, in the other two series 0.9 per cent solution was applied once a week. The production of tumours was not very different in the four series. 2. Tumours appeared in mice after painting with a 0.3 per cent benzene solution of 1: 2: 5: 6-dibenzanthracene applied twice a week for sixteen weeks but not in mice painted for only eight weeks. 3. Tumours were well advanced after thirty weeks in mice painted biweekly with 1: 2: 5: 6-dibenzanthracene solution, but if the painting was stopped after sixteen weeks tumours did not even appear until the thirty-eighth week of the experiment. 4. Tests for hydrocarbon were made on the subcutaneous tissues of rats at the site of injection of 1: 2: 5: 6-dibenzanthracene and of 1: 2-benzanthracene in solution in lard. The fluorescence spectrum was used, which under the conditions of these experiments is capable of detecting about mg. hydrocarbon in 1 C.c. of the benzene extract of dried rat tissue. The very slightly carcinogenic hydrocarbon 1:2-benzanthracene is eliminated, or destroyed, more easily than is the carcinogenic 1: 2: 5: 6-dibenzanthracene. 5. Mice were painted with a benzene solution of 1: 2: 5: 6-dibenzanthracene, and other series with a solution of 1: 2-benzanthracene of the same concentration. The hydrocarbons could not be detected in the skin four weeks after the last application whether the mice were painted biweekly for one week or for thirty-two weeks.

8 MECHANISM OF CARCINOGENESIS BY CHEMICAL COMPOUNDS Tumours developed in 2 mice from a series of 10 painted for eight weeks with a 0.3 per cent benzene solution of 1: 2-benzpyrene and then for eight weeks with a similar solution of 1: 2: 5: 6-dibenzanthracene. No tumours appeared in a control group (A) painted for only eight weeks with 1:2-benzpyrene solution, nor in another control group (B) painted for only eight weeks with 1: 2: 5: 6-dibenzanthracene solution. NOTE: The author is indebted to the British Empire Cancer Campaign for the grant under which this work was carried out. REFERENCES 1. COOK, ]. W., RIEGER, I., KE:lINAWAY, E. L., ASD MAYNEORD, \V. V.: Proc. Roy. Soc. B. 111: 455, SHEAR, M. J.: Am. J. Cancer 26: 322, CHALMERS, J. G.: Biochem. J. 28: 1214, BERENBLUM, 1., AND KENDAL, L. P.: Biochem. J. 30: 429, RIEGER, I.: Ph.D. Thesis, University of London, BOYLAND, E.: Biochem. J. 27: 791, 1933.