(From the Cancer Research Laboratories, University of Manchester)

Transcription

1 COMPARATIVE ACTIVITY OF SOME CARCINOGENIC HYDROCARBONS J. M. TU ORT AND C. C. TWORT (From the Cancer Research Laboratories, University of Manchester) During recent years an assortment of experiments has been published on the relative activity of synthetic carcinogenic hydrocarbons. A record of our own experience with : 2 : 5 : 6-dibenzanthracene, 3 :4-benzpyrene, and methylcholanthrene may be useful as an addition to those already appearing in the literature. Varying dilutions of these substances in chloroform were applied daily to the skin of mice, as well as dilutions in other solvents such as oleic acid. TABLE I : Carcinogenic Potency of Three Hydrocarbons in Chloroform Agent* Nutior 3er cent Potency Total Tumours Tumours Week When ill Survivorc Showed Growth Percentage at 40 Weeks Percentage at 25 Weeks H. C _ I-I.C H. C H. C , * H. C. 7 = : 2 : 5 : 6-dibenzanthracene. H. C. = 3 : 4-benzpyrene. H. C. 9 = Methylcholanthrene. EXPERIMENTS WITH CHLOROFORM AS A SOLVENT Nine ordinary experiments in which chloroform was used as solvent were carried out, with mice in each. Dilutions of, 0.05 and per cent of each hydrocarbon were utilised. In addition two further experiments were performed with dibenzanthracene and benzpyrene in dilutions of 0.2 per cent. The experiments were prolonged for forty weeks where possible, the survivors then being killed. Some of the experiments with benzpyrene were repeated because of the high death rate of the animals. In these cases the mean value of the two experiments is given. Using our ordinary procedure for determining carcinogenic potency () we arrived at the figures given in Table I. Scrutiny of the table reveals that H. C. 7 is considerably less carcinogenic 0

2 COMPARATIVE ACTIVITY OF SOME CARCINOGENIC HYDROCARBONS than the other hydrocarbons, whether the potency figures or the percentages of malignant tumours at 25 weeks are used for comparison. H. C. appears to be at least five times as carcinogenic as H. C 7, and H. C. 9 more than twice as carcinogenic as H. C.. The H. C. 7 series of experiments, in which the death rate was low, gave results proportional to the strength of the agent. This is shown, not only by the potency figures, but also by the percentage of living animals which bore malignant tumours at the end of the experiment. The potencies of 9, 70, 60 and 30 approximate to a mathematical progression. The H. C. series also gave results proportional to the strength of the agent, the potency figures here being 535, 36, 237 and 5. With the three strongest dilutions all surviving animals bore malignant tumours at thirtythree, thirty-five and a half, and thirty-seven and a half weeks respectively, while the per cent dilution yielded only 30 per cent of malignant tumours by the end of the experiment (forty weeks). The H. C. 9 series gave potency Agent I H.C. 7 H. C. H. C. 9 TABLE I I : Efect of Three Hydrocarbons on the Spleen Per cent with Hyaline No. Per cent Large Degeneration I_ figures of 757,37, and 375, and per cent of malignant tumours at twentyseven, thirty-four, and thirty weeks respectively. These results are poor, owing to the high death rate among the animals, from various causes, for only 4 of the 300 survived at thirty-five weeks. But our results leave no doubt that methylcholanthrene is considerably more carcinogenic for the skin of mice than the other two hydrocarbons under test. Examination of Spleen: An examination of the spleens of all surviving ani-. mals which were killed, and many of those which died during the course of the experiments, revealed a significant difference in the effect, direct or indirect, of the three agents on this organ. An enlargement of the spleen, the presence of an unusual number of myeloid and polynuclear cells, and hydine changes, often in an advanced condition, were much more prevalent among the mice painted with H. C. and 9 than among those painted with H. C. 7. In Table I are given the percentage numbers of enlarged spleens, weighed in the killed animals and estimated in those which died; also, the percentage of mice whose spleens showed obvious hyaline degeneration. The spleens were designated large when they weighed over 350 mg. As shown in the table, splenic enlargement and hyaline changes were much more prevalent among the mice painted with methylcholanthrene or benzpyrene than among those painted with dibenzanthracene. Moreover, the fact that 54 per cent of the mice painted with H. C. 7 survived for 40 weeks, whereas only 0 per cent of those treated with H. C., and less than 2 per cent of those painted with H. C. 9 survived for this time, makes the results much more striking,

3 2 J. M. TWORT AND C. C. TWORT Type of Skin Tumours: An examination of the skin of the mice bearing malignant tumours revealed that H. C. 9 had somewhat less tendency to produce a non-keratinising type of malignant tumour of the round-cell or basalcell variety, but a greater tendency to produce the spindle-cell variety, than H. C. 7 and. Thus, of the malignant tumours induced by H. C. 7 in 45 mice, 0 were composed almost entirely of non-keratinising cells, and of these but 2 were of the spindle-cell variety. On the other hand, among 0 tumours induced by H. C., 7 were non-keratinising, and of these, 3 were spindlecelled; while among 50 tumours induced by H. C. 9 all 6 non-keratinising growths were composed almost entirely of spindle cells. Before we refer to some miscellaneous experiments carried out with H. C. 7 and in various non-volatile solvents, and while we are comparing the activity of the agents when dissolved in chloroform, it is convenient to give some figures relative to their activity when dissolved in oleic acid. Based on animal reaction, the comparative carcinogenic activity of some agents, dissolved in these two solvents, estimated from an analysis of all available relevant experiments, was very approximately as follows : Agent Chloroform Oleic Acid Chrysene... + Synthetic tar, fraction Synthetic tar, fraction Synthet.ic tar, fraction :2:5:6-dibenzanthracene....0.o 3 :4-benzpyrene o Methylcholanthrene Dibenzanthracene has been taken as the standard unit from which to compare in both series of experiments, but it must be understood that this agent is considerably more carcinogenic when dissolved in chloroform than when dissolved in oleic acid. It will be noted that benzpyrene and fractions 0 and 9 of the synthetic tar when dissolved in oleic acid show only about two-fifths of the activity to be expected from a consideration of their activity when dissolved in the volatile solvent. Thus benzpyrene, although five times as potent as dibenzanthracene in chloroform, is only about twice as strong in oleic acid. On the other hand, chrysene, which has an almost negligible activity in chloroform, has quite an appreciable carcinogenic potency when applied in oleic acid. The crystalline picrates of our synthetic tar, represented by fraction 32, were unfortunately not tested in oleic acid solution, but it is highly improbable that results comparable to those obtained with dibenzanthracene would have been forthcoming, although in chloroform the results with these two agents were assessed to be similar. As a matter of fact, our tar is now being subjected to molecular distillation, as it is felt that by this means the very active constituents of it may be isolated. EFFECT OF DIFFERENT SOLVENTS Experiments with solvents other than chloroform have so far not been carried out with H. C. 9. But as many as 34 experiments, each with mice, have been performed with H. C. 7 and dissolved in oleic acid, butyric acid,

4 COMPARATIVE ACTIVITY OF SOME CARCINOGENIC HYDROCARBONS 3 liquid paraffin, several petroleum oils of varying carcinogenic activity, shale oil, benzene, etc. In some, oleic and butyric acid or oleic and stearic acid were used as a mixed solvent. In others lanolin was added to the solvent or the animals were treated with lanolin after painting. In one 0.0 per cent calciferol was added to the solution of H. C. 7 in oleic acid and this appeared to increase carcinogenicity, as it did when added to mineral oils. Perhaps the most interesting of the findings which have arisen from these experiments is the confirmation of the sensitising effect of oleic acid. Several years ago we found that the activity of the lowly carcinogenic hydrocarbon, chrysene, appeared to be considerably increased when oleic acid was used as a solvent (2) instead of liquid paraffin or even benzene, and it was naturally of considerable interest to us to find that the carcinogenic activity of dibenzanthracene and benzpyrene was also affected. Although, as we have just seen, both benzpyrene and dibenzanthracene showed themselves to be somewhat more active for the skin when dissolved in a volatile solvent such as benzene or chloroform than when dissolved in oleic acid, their potencies were much further decreased on solution in liquid paraffin. For example,. in four experiments with H. C. 7 in which 0.05 per cent of this hydrocarbon was dissolved in chloroform, oleic acid, a lowly carcinogenic mineral oil, and liquid paraffin, the final potency figures were 60, 22, 26 and 3.5 respectively. Table I gives the data relating to tumours and survival at the thirty-fifth week, for comparison : Solvent TABLE : Effect of Different Solvents Survivors Potency Total with Total Tumours Tumours Tun,ours Survivors Chloroform Oleic acid Mineral oil Liquid paraffin We can see from the table the very great difference in the activity of the hydrocarbon according to the nature of the solvent utilised. Oleic acid appears to act similarly to the mineral oil, but in these solvents the hydrocarbon fails to induce malignancy to the extent it did when dissolved in chloroform. It is obviously still less active when dissolved in liquid paraffin, as regards the production of both benign and malignant tumours. Oleic Acid and Other Organic Acid Solvents: Comparison of oleic acid with other organic acid solvents is of interest. For example, some experiments with per cent H. C. 7 in oleic acid, butyric acid, and oleic acid and 2.5 per cent stearic gave potencies of 45, +, and 45 respectively. When H. C. 7 was dissolved in equal parts oleic and butyric acid the resultant potency was 9. There were thus indications that butyric acid was lessening the activity of the H. C. 7 as much as liquid paraffin, but the effect of stearic acid appeared to be neutral. Actually the experiment with butyric acid yielded but one tumour throughout the experiment, although of the mice survived for forty weeks.

5 4 J. M. TWORT AND C. C. TWORT Experiments in which 5 to 0 per cent of the organic acids-oleic, butyric, stearic and lactic-were added to 0.2 per cent chloroform solution of H. C. gave results of no great significance except that the potencies appeared to be somewhat higher than the controls, more especially when oleic acid was added. Lanolin Experiments: Several experiments were performed with H. C. 7 in which either a small quantity of lanolin was added to the chloroform solution or the animals were painted with a mixture of lanolin and olive oil after the H. C. 7 applications. The potency of the H. C. 7 appeared to be increased somewhat in both cases. When, however, oleic acid replaced chloroform as a solvent the potency appeared to be somewhat lowered, but there was not SUEcient difference in the potency figures for the results to be of much significance. It must be borne in mind, however, that the action of lanolin may be very subtle. We have performed numerous experiments with lanolin in conjunction with carcinogenic agents of various types and strengths during the past ten years. The addition of lanolin or subsequent painting with it after applications of carcinogenic mineral oils invariably leads to a considerable lessening of tumour yield. This is also true when lanolin is applied after dilute solutions or small quantities of pure carcinogenic gas tars have been used for painting. If, however, relatively large quantities or strong solutions of gas tars are applied, subsequent painting with lanolin or addition of lanolin to the tar will result in a yield of tumours greater than that observed among the controls treated with tar only. TYPE OF AGENT AND ITS EFFECT ON LATENT PERIOD In a further very interesting series of experiments an attempt was made to determine the effect of various agents in retarding or accelerating the development of tumours of the skin, the cells of which by previous treatment with selected carcinogenic agents had been rendered abnormal. These cells may be presumed to be in all phases of cancerous reversibility and irreversibility, according to each individual s original intrinsic susceptibility to carcinogenic agents. In one series of experiments a 0.2 per cent solution of benzpyrene in chloroform was applied five times a week, for six weeks, to the backs of 500 mice. At this time a few animals developed tumours, and they were discarded. Of the remainder, four groups composed of mice were segregated in such a way that they resembled one another, as far as possible, in colour, sex, and weight. One of the four groups was set aside as controls, and received no further applications whatever, the others were painted five times a week, for thirty-five weeks, with oleic acid, liquid paraffin, and lanolin respectively. All the survivors were then killed. It will be seen from Table IV that neither subsequent painting with lanolin nor with liquid paraffin appeared to affect the yield of tumours to any appreciable extent. On the other hand, the subsequent application of oleic acid raised the tumour yield to a very marked degree. It might have been expected, in view of our previous knowledge of the prophylactic action of lanolin, that this substance would have retarded the development of a tumour from an irreversibly cancerous cell. -4s a matter of fact, however, our original experiments in this field showed that, although lanolin protects the normal skin from the noxious effects of our carcinogenic

6 COMPARATIVE ACTIVITY OF SOME CARCINOGENIC HYDROCARBONS 5 TABLE JV: Effect of Various Agents on Deveelopment of Skin Tumour Total Survivors at Agent Potency Tumours Tumours 35th Week Nil (Controls) Liquid paraffin Lanolin Oleic acid agents, it will not, at least in mice, delay the advent of malignancy of a tumour once it has made its appearance. It is not surprising, therefore, that in this instance the irreversibly cancerous cells were unaffected by the lanolin, the tumour lag being similar to that of the control group of animals. It was interesting to note that in a parallel series of experiments where a pinene synthetic tar was substituted for benzpyrene, the yield of tumours by the oleic-acid treated animals was not very different than that of those treated with other agents. This is in conformity with previous observations, oleic acid showing itself to be a better sensitizer for a single relatively weak carcinogenic hydrocarbon than for a mixture of carcinogenic and non-carcinogenic hydrocarbons such as is present in our synthetic tars. This difference is probably to be accounted for by sensitization of the dermatitic inhibitory constituents of the tar. CONCLUSIONS. Dissolved in chloroform, 3 :4-benzpyrene is about five times as carcinogenic as : 2 : 5 : 6-dibenzanthracene. 2. Methylcholanthrene is about twice as carcinogenic as benzpyrene. 3. The crystalline picrates of a pinene synthetic tar made at 50" C. were about as carcinogenic as dibenzanthracene. 4. The frequency of hyaline degeneration and enlargement of the spleen is related to the carcinogenicity of the agent applied. 5. The frequency of spindle-celi epitheliomas is also, apparently, related to the carcinogenic activity of the agent. 6. Cells rendered irreversibly cancerous by benzpyrene are not retarded by lanolin from developing into a tumour. 7. Cells rendered abnormal by a few applications of benzpyrene quickly pass into the irreversibly cancerous phase when stimulated by oleic acid. Liquid paraffin, lanolin, and some other agents apparently have no such action. REFERENCES. TWORT, C. C., AND J. M.: Am. J. Cancer 7: 293, TWORT, C. C., AND BOTTOMLEY, A. C.: Lancet 2: 776, 932.