FURTHER OBSERVATIONS ON THE ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS

Transcription

1 FURTHER OBSERVATIONS ON THE ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS J. M. TWORT AEFD C. C. TWORT (From the Laboratories of the Manchester Conamittee on Cancer, England) The study of the effect on the internal organs of mice of the application of carcinogenic agents to the skin has occupied our attention during recent years, a preliminary analysis of findings in several thousand animals having already been published (1). Most of these earlier experiments were performed with the primary object of testing the reaction of the skin to various carcinogenic agents. More recently a number of experiments have been carried out specifically for the purpose of correlating any changes observed 'in the skin with differences in the morphology of the internal organs. This paper will be devoted for the most part to a description of our findings in these special experiments. Having found that the general health of the animal has an important bearing on tumour incidence, we were led to believe that the " balance " of certain organs might play some part in the cancerous process. We therefore from time to time weighed or measured certain of the internal organs of large batches of animals, and from the results obtained have been able to compile correlation values for the various organs one with another, and to compare the organs as to actual size or weight. By this means we have been able to compare (1) tumour and tumourless animals; (2) animals bearing malignant tumours and those without such tumours; (3) painted and control animals; (4) males and females in the different groups. The following organs were weighed: spleen, brain, and pituitary gland of certain groups. The following organs were measured: pituitary gland, thyroid, and parathyroid of certain groups. The body weight of each animal was recorded. The pituitary glands were measured in the fresh state, under a low power of the microscope, while stained sections of the thyroid and parathyroid were projected and measured with a planimeter. Correlation values for all animals were obtained for: brain and body weight, brain and spleen, brain and superficial area of pituitary, body weight and spleen, body weight and superficial area of pituitary, spleen and superficial area of pituitary. CORRELATION VALUES OF TUMOUR AND TUMOURLESS ANIMALS Here the relevant experiments, for purposes of analysis, were divided into two groups: (a) those in which the animals were killed in pairs during the course of the experiment; (b) those in which the animals were killed at the termination of the experiment. We shall first consider (a) those experiments in which the animals were killed in pairs. On a tumour being observed, the bearer was killed together 78

2 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 79 with a tumourless companion of the same sex, and as near as possible of similar colour and body weight. In two experiments with our pinene synthetic tar B/19, 400 males and 400 females were utilised; one experiment with shale oil comprised 200 females, and one with petroleum oil 50 males and 50 females. The animals were grouped in tens, and correlation values for spleen, brain, body weight, and the superficial area of the pituitary were calculated for the various tumour groups and corresponding tumourless groups. Only two groups were available from the animals painted with petroleum oil. Four groups of animals painted with synthetic tar B/19 and subsequently injected with oil were also utilised. Here, however, each group contained thirty mice. There were, in all, 820 animals, divided into thirtyseven paired groups for purposes of comparison. Contrary to expectations, there was a tendency for the correlation values to be higher in the tumour groups, this being especially noticeable among the animals painted with shale oil and petroleum oil. See Table I. TABLE I: Frequency with Which Correlation Values Were Greater in Agent B/19 Shale oil Petroleum oil (oil injected) TOTAL ', No. of Bodywt. Body Body Pitui Pitui Brain paired and wt.and wt.and taryand taryand and groups _ pituitary ~ ~ spleen brain spleen brain spleen When early and late tumour groups of the tarpainted animals were compared there was little indication that the early groups gave relatively higher values than the groups of animals which bore their tumours towards the end of the experiment, but in view of the difference in the correlation values for the various organs in individual groups, it is possible that the number of animals was not sufficient to determine this point. (b) On the other hand, correlation values obtained from animals killed at the end of numerous other experiments gave, on the whole, some indication that tumour animals were better " balanced." The results obtained from an analysis of 200 animals painted with petroleum oils and 434 painted with dibenzanthracene or gas tar were as follows: Body wt. Body wt. Body wt. Pituitary Pituitary Brain and and and and and and No. of paired groups pituitary spleen brain spleen brain spleen Petroleum oils Gas tars, etc Here again the figures indicate the frequency with which the correlation values were greater in the tumourbearing animals than in the tumourless. It is to be noted that they are again in favour of the tumourbearing animals. It appears probable from these results that animals which are highly

3 80 J. M. TWORT AND C. C. TWORT sensitive to carcinogenic agents are, on the whole, better balanced in respect of pituitary, spleen, etc., than less sensitive animals, This excess correlation of the organs of sensitive animals over and above those of less sensitive animals seems to be lost after they have borne their tumours for some time, for when groups of mice bearing malignant tumours were compared with those bearing only benign tumours, lower correlation values were usually found in the former. This lowering of balance values among animals with malignant tumours is presumably the indirect effect of the tutnour. In order that the reader may gain an idea of the extent of the correlation of the different organs we have compiled Table 11, our values being obtained TABLE I1 : Correlation Values Given by Painted Animals and Unpainted Controls I I I Body Body Body Brain Brain wt. and wt. Wt. and pitui and pituiand and tary spleen brain tary spleen 237 epitheliomas 490 benign tumours 458 hyperplasia 578 controls Spleen and pituitary Average of six correlations by the simple ranking method where perfect correlation equals unity. The experiments were performed with gas tars, synthetic tars, and pure carcinogenic hydrocarbons. Altogether 1763 mice were available, the animals, for convenience, being separated into groups according to colour and sex. It will be noted in this table that the control values tend to be the highest and the malignant group the lowest. As a matter of fact, with these animals the malignant group correlation values in no instance took premier position. It will be seen that the highest correlation value in each group is given by the body weight and brain or the body weight and pituitary area. A special point to be noted is that the average correlation value for body weight and spleen was very low in the hyperplasia group. The reason for this is that abnormal animals with very small spleens are more frequently encountered among those which show only a hyperplastic condition of the skin than in those which bear tumours, a small spleen indicating lack of vigour, and consequently a lack of response to the carcinogenic agent. A few such animals may appreciably affect the correlation of the spleen with other organs. A series of animals which were painted for a relatively short time may also be considered here. A few years ago, when the effect of carcinogenic agents and their watersoluble derivatives on bacteria, protozoa, seeds, etc., was being tested, some experiments were also performed to determine whether the application of small quantities of such agents to the skin of mice would have a stimulating action on the animal. About 300 mice of both sexes were painted daily with a small quantity of a 1 per cent solution of our synthetic tar, B/19, and some 300 unpainted animals of approximately the same weight were kept as controls. The animals were killed in batches, mostly before the tumourbearing period, i.e., at the fourth and eighth week, and were compared

4 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 81 with the controls. About 400 animals were available at the end of the experiment. Body weight was utilised in the main as a criterion of stimulation, but the result was negative, there being a certain amount of debilitating action on the part of the tar. We find here that there was also a tendency for correlation values to be lower in the painted animals. The animals were correlated in tens, the average correlations obtained for the various organs in both groups being as follows: Body wt. Body wt. Body wt. Brain Brain Spleen and and and and and and Animals pituitary spleen brain pituitary spleen pituitary Painted Controls It will be noted that body weight and brain, and body weight and pituitary, again give the best values. To sum up it may be said that correlation values obtained by consideration of the gross size of the organs leads us to infer that balance values, especially as regards the pituitary gland and body weight, are usually higher in animals which bear tumours, as compared with these that are relatively less sensitive to carcinogenic agents, but that when the tumours of the former animals show advanced malignancy this balance is lowered. Control, unpainted animals, however, tend to give the highest correlation values, the painting apparently upsetting balance. SEX DIFFERENCES For relative correlation purposes about 1200 females and 900 males, divided into twentysix paired groups, were utilised. The frequency with which correlation values for each group of organs was higher in the female than in the male was as follows: No. of Body wt. Body wt. Body wt. Brain Brain Sfileen paired and and and and and and groups pituitary spleen brain pituitary spleen pituitary The average correlation values for the various organs were: Body wt. Body wt. Body wt. Brain Brain Spleen and and and and and and Sex pituitary spleen brain pituitary spleen pituitary F M Here again we see that body weight and brain, and body weight and pituitary, give the best values. Also it will be noticed that only in the correlation of pituitary gland and spleen are the female groups more often below than above the corresponding male groups, and it is only here that the actual average correlation values for the female groups are lower. From a consideration of these findings and those relating to tumour and nontumourbearing animals one might expect females to be slightly more

5 00 h3 Agent /l9 /l9 /l9 119 Shale oil Petroleum oil (injected oil) TABLE 111: Comparison of Tumour and Turnourless Animals of Approximately Equal Body Weight Experiment no. hto. of animals Pituitary area (sq. mm.) Spleen (mg. Brain (mg.) C.S. 53 AD 60 tumour 60 tumourless C.S. 53 AD 90 tumour 90 tumourless C.S. 52 AD 67 tumour 67 tumourless C.S. 52 AD 76 tumour 76 tumourless C.A. 37 A and B 40 tumour 40 tumourless C.R tumour 10 turnourless C.L. 56 A and B 60 tumour 60 tumourless No. of animals Pituitary dry wt. (mg.)

6 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 83 sensitive to carcinogenic agents than males. We have not studied this question fully, but a preliminary examination of thirty experiments in which 3000 animals were utilised gave the following order of decreasing sensitivity in the colour and sex groups. Benign tumour Malignant turnour Albinoeye female Albinoeye female Pigmentedeye male Albinoeye male Albinoeye male Pigmentedeye male Pigmentedeye female Pigmentedeye female Here each experiment was treated separately, and the average time elapsing before the members of each of the four groups developed a tumour was calculated, benign and malignant tumours being considered separately. The average time was then converted to a percentage basis with a mean time period of 25, this procedure being necessary to bring experiments with agents of variable carcinogenic potency into line and thereby allowing the mean given by each group in the thirty experiments to be available for comparison. It will be seen that the differences are slight and also that the presence or absence of pigment seems to be a more important factor than sex (2). SIZE OF ORGANS AND TUMOUR DEVELOPMENT We now come to a consideration of the actual size or weight of the organs. First, we shall consider their possible bearing on tumour development. Here again the experiments are conveniently divided into two parts: (a) those in which the animals were killed in pairs during the experiment; (b) those in which the animals were killed at the termination of the experiment. (a) In Table 111 are given our findings in the three experiments with tar B,/19 in which the animals were killed in pairs, one tumourbearing and one tumourless. A similar experiment with petroleum oil and one with shale oil are also included in the table. The only points of interest here are that in six out of the seven groups the tumour animals have a larger average pituitary gland than the tumourless animals, while there is a tendency for the brain, spleen, and body weight of the former to be smaller. In experiments where sufficient material was available, body weight and colour were ignored, and tumour and tumourless animals with equal brain weights were paired and their organs compared. It will be seen from the results given in Table IV that the pituitary area is larger in all tumour groups, the spleen is larger in four out of the five groups, the body weight in three out of the five; in one of the three instances the pituitary dry weight was slightly less. Moreover, when the animals considered in these experiments were paired more accurately according to body weight, date of killing or colour being ignored, the same result was obtained, the tumourbearing animals having, on the whole, a somewhat smaller brain, a larger pituitary gland, and a somewhat larger spleen. Sectional areas of the thyroid and parathyroid glands of of these mice were determined by means of a projection apparatus and planimeter. We were able to compare five groups of tumourbearing animals with five groups

7 Agent /l9 00 /l9 #A Shale oil /l9 /l9 Experiment no. C.S. 53 AD C.S. 53 AD C.S. 37 A and B C.S. 52 AD C.S. 52 AD TABLE I\': Comparison of Turnour and Turnourless Animals of Equal Brain Weight No. of animals 40 tumour 40 tumourless Sex hlale Body wt. (gm Pituitary area G xy) (sq. mm.) Spleen (mg.) tumour 40 tumourless tumour 25 tumourless tumour 40 tumourless tumour 70 tumourless No. of animals Pituitary dry wt. (mg.) I

8 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 85 TABLE V: Comparison of the Thyroids and Parathyroids of Tumour and Tumourless Animals Agent /19 Experiment No. C.S. 53 AD No. of Animals 17 tumour 22 tumourless Sex Thyroid (sq. mm.) Parathyroid (sq. mm.) /19 C.S. 53 AD 32 tumour 24 tumourless 1.(I /l9 C.S. 52 AD 22 tumour 26 tumourless /19 C.S. 52 AD 27 tumour 28 tumourless Shale oil C.A. 37 A and B 12 tumour 17 turnourless Total 110 tumour 119 tumourless and fen and fen which did not bear tumours (Table V). Actually four out of the five tumourbearing groups gave average larger sectional areas of the parathyroid than the tumourless, but in four out of the five groups the average sectional areas of the thyroid were smaller in the tumour animals. Some previous researches gave indications that the percentage of tumours was higher in mice where the parathyroid was present in a single section of the thyroid gland than in those in which it was absent (1). The differences observed between tumour and tumourless mice when the animals were killed in pairs during the course of the experiment were naturally not so clearly demonstrable among animals killed at the end of experiments, many of which had had their tumours for some considerable time. Thus among mice killed at the end of experiments with tars there was little indication that tumour animals had larger pituitary glands or heavier spleens than those which were tumourless, but where definite malignancy was present the spleen was, of course, as a rule enlarged. Nevertheless, mice painted with petroleum oils again showed average differences between the tumour bearers and those with only hyperplastic skin, as will be seen from Table VI. Here in all four tumour groups the spleen weight and pituitary area are above the average given by the four tumourless groups, although the body weight and brain weight are on an average higher in the latter animals. Eight experiments with dilutions of dibenzanthracene form the basis for Table VII, females only being considered, as there were relatively few males in the experiments. All the animals surviving to the end of the experiment were killed, i.e., at the fortieth week. They are divided into three groups: (1) having frankly malignant skin tumours, (2) having benign tumours or tumours showing only early malignancy, (3) without tumours. The animals with malignant tumours showed a considerable increase in size of the spleen and a considerable decrease in body weight. The average

9 J. M. TWORT AND C. C. TWORT TABLE VI : Comfiarison of the Organs of Tumour and Tumourless Animals Killed at the Termination of Experiments Agent Petroleum oils: Batch 1 No. of animals 20 tumour 62 tuinourless Sex Body wt. (gin.) Pituitary area (fxy) sq. mm.) Spleen (mg tumour 48 tumourless Petroleum oils: Batch 2 10 tumour 20 tuinourless tunlour 12 tumourless TOTAL Batches 1 and 2 30 tumour 82 tuinourless Batches 1 and 2 27 tumour 60 tumourless Skin TABLE VII; Comparison of the Organs of Tumour and Turnourless Animals Painted with Dibenzanthracene 1 I 1 No. Body Pituitary wt. area (+ xy) anirnals of hi.) (sq. mm.) 1 ii:;. ~ 1 Malignant tumour ,772 Benign tunlour H yperplasia Spleen (mg.) Brain (mg.) superficial area of the pituitary was also lower than in the other two groups. The differences in the benign tumour and hyperplastic groups are insignificant. From the mice considered in Table VII there were selected animals from each group which had the same brain weight, and the organs were compared, i.e., (1) an animal among those in the benign group was paired with one having a similar brain weight in the malignant tumour group; (2) an animal among those of the benign group was paired with one of similar brain weight in the hyperplastic group; (3) an animal among those in the malignant group was paired with one of similar brain weight in the hyperplastic group. The results are given in Table VIII, wherein the number of times the organs are heavier or the superficial area of the pituitary is larger is given under the various organs for each group compared. Here again there is a considerable difference between the malignant and the other two groups as regards body weight and weight of the spleen but no significant differences appear between the animals bearing benign tumours and those which show only hyperplastic skins, except possibly as regards body weight. The superficial area of the pituitary among these animals shows no significant differences.

10 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 87 TABLE VIII: Comparison of Tumour and Tumourless Animals of Equal Brain Weight (The figures show the frequency with which the body weight and the organs are heavier or the superficial area of the pituitary is larger) No. of pairs Skin Spleen Body wt. I Pituitary area Malignant tumour Benign tumour Malignant tumour H yperplasia I Benign tumour H y perplasia TABLE IX: Comparison of the Pituitary Weight of Tumour and Tumourless Animals NO. of pairs I Skin 1 Pituitary dry weight Malignant tumour Benign tumour Malignant tumour H yperplasia Benign tumour H yperplasia mg mg mg mg nig mg. The dry weight of the pituitary gland of some of the above animals was determined and the average dry weight compared in the different groups, the animals again being paired according to brain weight. The results given in Table IX show that the pituitary dry weight of the animals bearing malignant tumours is on an average lower than that of animals of the benign and hyperplastic groups, while that of the hyperplastic group is below that of the benign group. This is further evidence that, on an average, mice bearing malignant tumours show not only loss of body weight and increase of splenic weight, but also possibly to some extent loss of pituitary weight, The number of animals available, however, was obviously too small to warrant definite conclusions from Table IX alone. SEX AND SIZE OF ORGANS Early in our investigations it was noticed that the size of the pituitary gland, as determined by a measurement of the approximate superficial area of its anterior surface, was on an average larger in female than in male mice, and this led us to a comparative investigation of other organs. We have already seen that correlation values for the organs of the females are, on the whole, higher than those of the male. In Table X are given the results obtained from an analysis of 15 paired groups of male and female mice, comprising 1214 males and 1578 females. As far as possible the paired groups were made up from animals of the same colour and which had been exposed to the same experimental conditions.

11 ~ ~ J. M. TWORT AND C. C. TWORT TABLE X: Sex in Relation to Size of Organs Group and sex 1. NO. if animals Agent Skin Tuniour Tumour 22.5 Pituitary area (sq. n1m. ) Mde Tuinourless Tumourless Tuinour Tumour Tumourless Tuniourless Feinale Tuinour and tumourles Tuniour and tuinourles Tumour and tuniourles Tuinour and tuiiiourles Tumour and tumourles Tumour and tumourles Tuinour and tumourles Tumour and tuniourles Tumour and tuniourles Tumour and tumourles Oils Oils Tuiiiour and tuniourles Tumour and tumourles Tuniourless Tumourless Fema le Tumour and tuniourles Tumour and tumourles Nil Nil Noriiial Normal I9 140 Nil Nil Normal Normal Nil Nil Normal Normal TOTAL: Comparing the fifteen groups we see: (1) that in every group the average body weight of the female is below that of the male; (2) that in eleven of the groups the superficial area of the pituitary is larger in the female; (3) that in eight of the groups the spleen is heavier in the female, and (4) that in

12 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 89 twelve of the groups the brain is heavier in the female. These we consider as rather remarkable findings. That the differences between the two sexes are not related to the agents applied to the skin is indicated by the three control unpainted groups: in the three female sections the average brain weight and superficial area of the pituitary are above the averages for the corresponding male sections, the spleen in one instance being slightly below. The averages for all the animals are compared at the end of the table, where it will be seen that the average body weight of the female is 1.3 gm. Body weight (gm Pituitary area (sq. mm.) GRAPH I. COMPARISON OF AREA OF PITUITARY (1s SQ. MM.) IN MALE AND FEMALE MICE Approximately 2000 killed animals. lower than that of the male, whereas the superficial area of the pituitary and the weight of the spleen and brain are above. The findings indicate that, although a' female mouse has usually a larger pituitary gland than a male of the same body weight, it has not usually a larger pituitary gland than a male mouse with the same brain weightin other words the female has usually a larger pituitary gland than the male of the same weight because it usually has a larger brain. The two accompanying graphs, compiled from about 2,000 animals, illustrate well the points under discussion, In general terms

13 90 J. M. TWORT AND C. C. TWORT it appears that the female mouse having a brain and pituitary gland similar in size to a male mouse will have a body weight of approximately 2 gm. less. It will be appreciated that a knowledge of these facts is essential for interpreting variations in a mixed group of tumour and tumourless animals, etc. Careful study of Table X shows that, owing to the great variation in individual animals as regards relative size or weight of their organs, it may be necessary to deal with very large numbers before one can arrive at definite Body weight (gm.) BRAIN Weight 360 of brain (mg.1 GRAPH 11. WEIGHT OF BRAIN IN MALE AND FEMALE MICE Approximately 2000 killed animals. conclusions. Thus in group 9, although this consisted of 273 animals, the results were not very convincing. Again, if male groups 1 and 2 are compared with 3 and 4 it will be noticed that, although the average body weights are slightly lower in the former, the average weight of the spleen and brain are appreciably higher. This is presumably not due to differences in strain! for the females of groups 1, 2 and 3,4 gave normal results, and all four groups received applications of the same agent for a similar period of time.

14 ORGANS OF MICE PAINTED WITH CARCINOGENIC AGENTS 91 EFFECT OF THE CARCINOGENIC AGENT ON THE PITUITARY GLAND In the first instance several batches of animals painted with synthetic tar, petroleum oil, and shale oil, were utilised as a source of pituitary glands. Here it was found that when the animals were divided into classes according to weight and sex, both average superficial area of pituitary and dry weight of pituitary were much the highest in the group of mice treated with shale oil and much the lowest in the groups treated with synthetic tar. It was considered that the differences were probably much too great to be explained by any differences in the nature of the substances applied, and a further experiment with shale oil tended to confirm this, the shaletreated animals.giving no pituitary enlargement over and above that of control animals. Similar results were found on the examination of the pituitary glands of several thousand mice previously painted with petroleum oils or synthetic tars. But it must be remembered that the experiments with petroleum oils were usually of much longer duration than those with tars, so that the age factor may have approximately balanced any differences due to other causes. Some more recent experiments with oils, tar, and dibenzanthracene, however, seem to give indications that the nature of the substance applied may have some influence on the pituitary gland. In 47 groups of mice compared, of which 13 were painted with oils, the ratio of pituitary weight and brain weight was appreciably higher in seven of the oil groups compared with the groups painted with tar and dibenzanthracene. Two groups of old control unpainted mice gave the smallest ratio. The results of the analysis of these animals are shown in Table XI. TABLE XI : Comparison of the Effect of Carcinogenic Agents on Size of Organs No. of animals Agent Pituitary Pituitary Pituitary Shale oil Petroleum oils Gas tars Dibenzanthracene Old control animals As regards the effect of age, it will be seen that the group of control animals mentioned in Table XI, which were about two years old, had pituitary glands on an average appreciably smaller than those of the painted animals included in the table, the latter being under eighteen months. Age, however, does not account for the marked differences we have sometimes found between shaletreated and tartreated animals, and it is possible that some unforeseen factor or factors are partly responsible for these differences. DISCWSSION Correlation values for total body weight, splenic weight, brain weight and pituitary area have been determined one with another for nearly 2000 animals. Correlation values for pituitary dry weights have also been determined for

15 92 J. M. TWORT AND C. C. TWORT about 500 animals. We have compared the average values given for control mice and mice which have been painted with carcinogenic agents on the skin. We have compared, also, mice which have developed only a hyperplastic condition of the skin with those which have developed benign tumours as well as those which have developed frankly malignant tumours. Mice which have developed malignant tumours have been compared with those in which the tumours were benign, and lastly males have been compared with females. Apart from correlations, the average size of the organs of tumour and tumourless animals, etc., has been compared. We have seen that even when dealing with relatively large groups of mice our results have on occasion been contradictory, so that we can sometimes speak only of indications rather than of definite conclusions. CONCLUSIONS 1. Correlation values for body weight, brain weight, splenic weight, etc., are usually higher for mice which develop tumours than those from the same experiment which do not develop tumours. Correlation values for mice which bear frankly malignant tumours are, however, usually lower than those given by the corresponding tumourless animals. 2. Correlation values are usually higher in females than in males for most of the organs considered. 3. Tumourbearing animals were found to have on an average a larger pituitary gland, a somewhat larger spleen, and a somewhat smaller brain than tumourless animals of the experiment having similar body weight. 4. The distinction between tumour and tumourless animals is best shown in those experiments in which the mice were painted with oils. This was true both as regards correlation values and actual size of organ. 5. Mice which bear tumours which show frank malignancy usually show a lowering of body weight and an increase of splenic weight. 6. mice have usually larger pituitary glands, heavier brains and, to a less extent, heavier spleens than males of the same weight. There are also indications that (1) Mice which bear tumours which show frank malignancy have usually a somewhat smaller pituitary area and weight than those in which the tumours remain benign. (2) Tumourbearing mice have usually larger parathyroid glands than those which bear no tumours. (3) The pituitary glands of mice exposed to different experimental conditions show on occasion greater variation than can be attributed to differences of strain, etc. (4) Mice over two years of age usually have pituitary glands below the average weight for adults. NOTE: In conclusion we should like to thank our laboratory assistant, Mr. Fred Dixon, for his valuable help in the practical work. REFERENCES 1. TWORT, J. M., AND TWORT, C. C.: Am. J. Cancer 23: 52, TWORT, C. C., AND TWORT, J. M.: J. Hyg. 32: 557, 1932.