Sex Difference in the Conjunctive Tumoral

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Sex Difference in the Conjunctive Tumoral Reaction of the Guinea Pig Towards Natural and Artificial Estrogens* Alexander Lipsch0tz, M.D., Luis Vargas, Jr., M.D., and Jorge Palma, M.D.

(From the Department of Experimental Medictne, Natzonal Health Service o/ the Republic o[ Chile, Santiago) (Received for publication May 2o, I94I)

INTRODUCTION The endocrine activity of the gonads is not respon- sible for this sex difference. We have reported that the The conjunctive tumoral reaction of the male guinea castrated male guinea pig reacts in a way similar to the pig towards esterified estradiol is different from that noncastrated male (6, 7)" One must then question of the female (7)- whether there is a sex difference in the metabolic fate Large spherical peritoneal fibroids are easily elicited in the of the follicular hormones which might explain the female guinea pig injected thrice weekly with 8o /zgm. or less sex difference in the tumoral reaction to estrogens. of estradiol 1 given as the benzoate in the course of 2 to 3 Comparative experiments with natural and artificial months (6, 9, io). Similar fibroids are not induced in the male under similar conditions. The tumoral seed on the spleen, on estrogens offer an opportunity to study this question the surrounding parts of the abdominal wall, and on the ventral experimentally. Artificial estrogens are more resistant surface of the stomach, and peritoneal fibrous strands are the against inactivation than the natural follicular hor- characteristic features of the conjunctive reaction in the male mones (4, i8). Therefore, there should be some dif- (6, 7). In the male the localization of the reaction on the left ference between the metabolic behavior in the organ- side is of great uniformity, especially at the beginning (I9). In contrast, the variation is enormous in the female (5, 9, io, 2x). ism of artificial and natural estrogens. If the sex The reaction in the male is probably due to a lesser susceptibility. difference in the conjunctive reaction towards follicular This is suggested by different facts. The left-sided tumoral seed hormones as established by our experiments were due typical of the male is found frequently in females treated with to some unknown sex difference in the metabolic fate srnallcr quantities of cstrogens (I, I5). An identical feature is of the hormones, one might question whether male observed in the female when the tumorigenic action of the follicular hormone is inhibited by other hormones such as or female guinea pigs would also react towards arti- testosterone, progesterone, or desoxycorticosterone (i2, I3). In- ficial estrogens in a different manner according to sex, dividual spherical fibroids appear also in the male when the as they do towards natural estrogens. injections of 80 /zgm. of estradiol benzoatc arc prolongcd for more than 4 months (7) or when similar quantities of the 17-caprylate or the x7-benzoate-3-n-butyrate ester of estradiol (5) EXPERIMENTS 2 which possess more potent tumorigenic power than the benzoic Thirty-seven male guinea pigs, weighing 235 to or dipropionic esters of estradiol (8) are used. And finally, the uriiformity of the reaction in the male becomes highly accentu- 680 gin., were injected with doses of stilbestrol or atcd when a smaller dose of the i7-caprylate as, for instance, hexestrol which in the female are sufficient to elicit Io /zgm. per injection is used (2), a dosage which in the female abdominal fibroids, or with doses four times greater. is sufficient to elicit large abdominal fibroids (6). The sex The results in the males were compared with those ob- difference of the conjunctive reaction was quite evident also tained in :z3 castrated females. The artificial estrogens in experiments in which tablets of free estradiol were used instead of injections of esterified estradiol (3). were given in olive oil; 0. 4 cc. containing 8o or 32o/~gm. The above facts have been established by our group on a of stilbestrol, and 4 o or i6o/zgm, of hexestrol respec- number of animals sufficiently large to reduce possibility of error. tively, were injected thrice weekly in the course of 87 Seventy-eight male and several hundred female guinea pigs were and 88 days (with the exception of 3 animals injected injected in the course of 69 to I4o days (2, 6, 7, I9) or treated with subcutaneous tablets (3). Full corroboration of our findings for 73 to 84 days only.) A total of 38 injections (3 to concerning a sex difference in the conjunctive tumoral reaction I2 mgm. of stilbestrol, and I. 5 to 6. 3 gm. of hexestrol) has been published by other workers (I7). was given. These timing conditions corresponded to those in our former work with the injection of free or * This investigation was aided by grants from The Jane Coffin esterified follicular hormones into males (6, 7)- Childs Memorial Fund for Medical Research, from Mr. Adolfo Eastman of Limache (Chile) and others. The results are summarized in Table I in which is 1 The artificial estrogens were generously supplied by Pro- given the number of animals which have developed fessor E. C. Dodds of the Courtauld Institute of Biochemistry, The Middlesex Hospital, University of London. 2 See thesis of J. Palma (i6).

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1941 American Association for Cancer Research. 576 Cancer Research extragenital tumors of a certain size; i.e., "class I," or on the abdominal wall and diaphragm in the vicinity tumors of a diameter not less than i mm., according of the spleen (Figs. 2 and 5). This left-sided orienta- to our classification (8, i i). It is evident from Table I tion of the tumoral seed on the stomach and abdominal that 38 injections of 8o /~gm. of stilbestrol or of 4 ~ wall may be present when the tumoral seed on the /,gm. of hexestrol given in the course of 3 months are spleen is lacking. In one animal injected with 32o unable to elicit tumors in the male whereas with the /,gin. of stilbestrol per injection the tumoral seed was same treatment a high incidence of extragenital fibroids found also on the intestine, especially on the cecum occurs in the female. Tumors appear in the male when and ascending colon (Fig. 6). This was the only quantities four times greater are given (Fig. 6). Even animal in which large tumors were present. The then the frequency of tumors in the male is very small tumoral seed on the intestine is a very rare localization. when compared with that in females treated with the In our previous experiments it occurred only after same quantity or with quantities four times smaller. injection of highly potent esters of estradiol in males With 320 /,gm. of stilbestrol or with I6o ~gm. of (Fig. 3). It may appear also in females (Fig. 4). hexestrol per injection there was only one animal with Frequently a tumoral seed can be seen also on the individual spherical tumors of class i or larger. Fig. 6 surface of the left kidney and not rarely on the right

TABLE I: EXTRAGENITAL TUMORS IN 37 MALE AND 2 3 FEMALE GUINEA PIGS; 36 TO 39 [NJ-ECTIONS OF ESTROGENS IN THREE MONTHS

Number of Number of animals with animals with Stilbestrol extragenital I{exestrol extragenital Sex of per tumors, per tumors, guinea injection Nunlber class I or injection Number class I * pigs #gm. injected larger #gm. injected or larger Females ...... 80 8 5 40 IO 5 Females ...... 8o 5 4

Males ...... 8o 9 o 40 Io o Males ...... 320 9 I 16o 9 I

9Class i tumors are not less than I ram. in diameter.

TABLE II: CONyUNCTIVE TUMORAL REACTIONS Or 37 MALt- GUINFA PIGS; 36 TO 38 IN~ECTIONS OF ESTROGENS IN THREE MONTHS

Number of animals with tumoral seed on the Number A ___ Of Abdominal animals #gin. Number Kidney . wall with per of ~v ~'~ fibrous injection animals Testicle Stomach Intestine Left Right" ~Left Right Spleen strands Stilbestrol ...... 8o 9 9 5 o 2 2 5 o 3 Stilbestrol ...... 32o 9 9 6 I* 6 2 9 o 8

Hexestrol ...... 40 I o 7 7 o 4 2 9 o 7 9 Hexestrol ...... 160 9 9 6 o I o 7 o 6 9

* See Fig. 6. shows the condition of the only male with tumors in one. Almost constant phenomena are small nodules the group injected with 320 /,gm. of stilbestrol. The on the albuginea of the testicle (Figs. 5 and 6). reaction in the only animal with tumors in the group Fibrous strands induced in the peritoneum by a receiving i6o /,gin. of hexestrol was less pronounced prolonged treatment with estrogens (22) almost never than this. failed to appear in our animals (Table II). They were As we observed in former experiments with natural more pronounced in the 32o and I6o /~gm. groups estrogens, absence of individual spherical tumors in than in the 80 and 4 ~ #gm. groups; but in the latter the male did not exclude other types of conjunctive groups the fibrous strands also were sometimes very proliferation. All the 37 males with but one exception conspicuous (Fig. 2). showed some sign of a conjunctive reaction. The re- suits are summarized in Table II. There was in most DISCUSSION of the animals a "tumoral seed"; i.e., an accumulation of small white nodules on the surface of different The results of our work with prolonged administra- organs. The localization of these nodules whose micro- tion of stitbestrol and hexestrol in guinea pigs leave scopical structure we have described (I4), was by no doubt as to the fact that with artificial estrogens preference on the left side--on the spleen, on the conjunctive proliferation is different in females and ventral surface of the stomach near the great curvature, males. There is the same sex difference as formerly

Extragenital fib~oids and tumoral seeds are indicated in solid black Fro. i.--Noncastrated female guinea pig--36 injections of 8o ~gm. of estradiol given as IT-benzoate in the course of 83 days (IV, 7J. Typical female reacnon. Numerous extragenital subserous tumors on the abdominal wall, diaphragm, stomach, omentum, pancreas, spleen, and liver. Tumors on the dorsal surface of the utcrus. FIa. 2.--Noncastrated male guinea pig--36 injections of 4o #gm. of hexestrol in the course of 83 clays (XVIII, 22). Typical male reaction. This may be observed also in tile female with small doses and very exceptionally with large ones. Tumoral seed on the spleen, stomach, abdominal wall, and diaphragm on the left. Adhesions bctwcen spleen and stomach. Fibrosis around kidneys, mesentery, and mesocolon highly pronounced. Small nodules on the tunica albuginea testis. Fro. 3.--Castrated male guinea pig--44 injections of 8o /~gm. of estradiol given as IT-caprylate in the course of lO 4 days (XVIII, I5). Exceptional type of male reaction combined with tumors as observed only with large doses of potent esters and (luring long treatment. Highly pronounced tumoral seed of typical left-sided localization. Fibrous strands of the mesentery and kidney. Fibrous cover on the cecum. Numerous subserous tumors on the smaller curvature of the stomach, pylorus, omentum, liver, right kidney, diaphragm, upper and under pole of the spleen. In the lower right: small nodule on the urinary bladder. Fro. 4.--Castrated female guinea pig--38 injections of 80 /*gm. of stilbestrol in the course of 87 clays (XVIII, 43). Typical female reaction, but combined with conspicuous tumoral seeds. Uterus (7.7 gm.) with numerous small subserous nodules and larger nodule between the horns. Apical tumor on the left horn. Several tumors in the omentum, in the mesentery, near the pancreas. Fibrous strands in the omentum. Tumoral seed on the spleen, stomach, and ascending colon. Fro. 5.--Noncastrated male guinea pig---38 injections of 80 >gm. of stilbestrol in the course of 87 days (XVIII, 6). Typical male reaction. Tumoral seed on the spleen, stomach, and abdominal wall on the left. Fibrous strands in the mesentery and on the upper part of the kidney. Small nodules on the tunica albuginea tcstis. FIG. 6.--Male guinea pig--38 injections of 320 /,gm. of stilbestrol in the course of 88 days (XVIII, 17), the only animal out of i8 which showed tumors. Exceptional type of male reaction--combination with tumors. Tumoral seed on the spleen, stomach, abdominal wall, diaphragm, and kidney extending to the right as seen especially on the stomach, and to the cecum and ascending colon. Fibrous patches, partly pedunculated , on the surface of the stomach. Cystic condition of the left kidney; nodules on the surface of the cyst which belongs entirely to the capsule of the kidney. Adhesions between spleen and stomach. Fibrous strands in the mesentery. Multiple tumors near the stomach and duodenum and between the spleen and pancreas. Small nodules on the tunica albuginea testis.

observed with natural estrogens. The tumoral seed SUMMARY especially on the spleen and the surrounding parts of A comparative study of the conjunctive tumoral the abdominal cavity and fibrous strands in the mesen- reaction towards prolonged treatment with artificial tery and mesocolon exhibits the typical pattern elicited estrogens (stilbestrol, hexestrot) has been made in in the male. Indeed we must emphasize, as we have female and male guinea pigs. already insisted above, that a similar reaction may also As with follicular hormones there was a conspicuous be seen in the female, especially with a small dosage sex difference in the reactive behavior toward artificial of estrogens. On the other hand, the individual spheri- estrogens. cal tumors of a certain size typical of the female may be seen also in the male, especially after large dosage Quantities of stilbestrol or hexestrol sufficient to elicit of estrogens. The difference in reaction between in the female a high frequency of large spherical females and males, however, was as pronounced with extragenital peritoneal fibroids, induce in the male artificial estrogens as with the natural follicular hor- only a tumoral seed on the spleen, the stomach, and mones. This is shown by comparison of Figs. 2 and 5 the surrounding left-sided parts of the peritoneum, with Figs. I and 4. and peritoneal fibrous strands. Since the metabolic fate of artificial estrogens is not With quantities four times greater, the tumoral seed identical with that of the natural follicular hormones and the fibrous strands become more conspicuous and our findings suggest that the sex difference in the con- only very exceptionally may individual spherical tu- junctive tumoral reaction towards estrogens, natural mors appear in the male in contrast to those observed or artificial, does not depend upon a sex difference in in the female. the metabolism of estrogens. There must be some This gives evidence that in respect to the sex dif- sex-linked factor which determines a sex difference in ference in the conjunctive tumoral reaction towards the susceptibility of the tissues for the proliferative estrogens natural follicular hormones and artificial action of estrogens and one may tentatively suggest estrogens behave in a similar way. that this factor is also of endocrine origin, probably Since the metabolic conditions of natural and arti- deriving from the anterior hypophysis. ficial estrogens in the body are dissimilar, our findings The difference in the tumoral reaction of females suggest that the sex difference in the tumoral reaction and males as described by our group can be interpreted is not due to metabolism of hormones differing accord- as a quantitative one. The male type of reaction ing to sex but to some other sex-linked factor which (tumoral seed on the left side of the body) is that of determines a different susceptibility of the reacting a female with an exceptionally low susceptibility; with tissue. It is tentatively suggested that this factor may larger doses tumors may appear also in the male be hormonal, probably originating in the anterior (Figs. 3 and 6). Though all of this is certainly in hypophysis. favor of a quantitative interpretation the following The fact that the male pattern of reaction is observed fact is somewhat contrary to our purely quantitative also in the female with a lower dosage of the estrogen, conception: in rare individuals, spherical tumors ap- and the female pattern is observed in the male with pear also in the male (Fig. 6), while the male pattern a higher dosage, is in favor of a quantitative interpre- of reaction is never extinguished. The individual tation of the described sex difference in the tumoral spherical tumors are so to say superimposed on the reaction. male pattern of reaction. Another feature, however, is again in favor of the quantitative interpretation: REFERENCES together with individual tumors appearing in the male I. BELLOLIO, P. Estudio comparativo sobre la accidn histero- the left-sided tumoral seed may extend to the right as trdfica y tumorlgena del benzoato y dipropionato de a consequence of injection of larger doses (6, 7). estradiol. Public. Med. Exper. (Chile), 2:1939. 2. CHAUME, J. Nuevos estudios experimentales sobre la espe- There is evidently a graded reactivity of the left and cificidad sexual en la reaccidn tumoral conjuntiva provo- right side of the body towards estrogens. This graded cada pot substancias estr6gcnas. Rev. chilena de hig. y reactivity of left and right belongs also to the female med. prey., 3:75-82. i94o-I94I. as has been shown by our observations in hundreds 3- CHAUME, J., and L. VARCAs. Estudio comparativo de la tumorig6nesis conjuntiva experimental del cuy con tabletas of experiments (9, ~o, :22). We are as yet unable to de estradiol subcutfineas e intraperitoneales. Rev. chilena explain this remarkable phenomenon. The spleen is de hig. y reed. prey., 3:83-89. 194o-1941. a most important starting point of conjunctive peri- 4. DINGEMANSE, E., and R. TvsLowrrz. Urinary Elimination toneal tumorigenesis elicited by estrogens. Whether of Estrogens Injected in Dogs. Endocrinology, 28:450- this means that the spleen has something to do with 457- 1941. 5- ICLES~AS, R. Tumores experimentales uterinos y extra- the intimate dynamics of this type of tumorigenesis genitales provocados por el benzoato de estradiol. Public. is indeed as yet not certain (2% 2I). Med. Exper. (Chile), I:I938.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1941 American Association for Cancer Research. Sex Difference in the Conjunctive Tumoral Reaction of the Guinea Pig Towards Natural and Artificial Estrogens

Alexander Lipschütz, Luis Vargas, Jr. and Jorge Palma

Cancer Res 1941;1:575-579.

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