THE EFFECT OF HUMAN AND ANIMAL URINE AND OF SERA UPON THE OVARIES OF ANIMALS

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1 THE EFFECT OF HUMAN AND ANIMAL URINE AND OF SERA UPON THE OVARIES OF ANIMALS ALBERT STRICKLER (Frm the Cancer Research Labratries, The Skin and Cancer Hspital f Philadelphia> The experimental results here recrded have as their basis the ability t determine scientifically the prductin f sex hrmnes by the human bdy and t measure accurately the amunts present in the bld, in varius rgans, and in the excreta, especially the urine. HORMONES IN HEALTH In health the excretin f estrin begins at puberty and under nrmal cnditins is cntinued until sme time after the nset f the menpause. Aschheim and Zndek () and Margaret Smith (), wrking independently, were respnsible fr the discvery that large quantities f estrin are excreted in the urine during pregnancy. The urine f the new-brn child has been fund t cntain this hrmne up t the furth day after birth; while small quantities have been recgnized in the urine f healthy males (Laqueur et al., 3; Fee et ai., 4). The gnadtrpic hrmne is knwn t be excreted during the greater part f the human life cycle. It has been fund in cmparatively large amunts in the urine in the first few days after birth; its presence has been determined in the urine befre the age f puberty (Seken, 5); during pregnancy a very marked increase in its excretin has been nted; and it cntinues t be excreted lng after the nset f the menpause (Osterreicher, 6). HORMONES IN RELATION TO MALIGNANT GROWTH The apparent relatinship between the gnadtrpic and sex hrmnes and certain types f neplasm, bth in man and in certain f the experimental animals, is a fact the full significance f which is nt understd. The presence f large quantities f the gnadtrpic hrmne in the urine f patients suffering frm hydatidifrm mle and frm chrinepithelima is a mst striking finding. Equally imprtant is the bservatin f Zndek that 8.5 per cent f a series f patients with carcinma f the cervix als shwed the presence f this hrmne, but in lesser amunt. Malignant tumrs f the testicle, i:e., teratma testis (Zndek, 7) and testicular chrinepithelima (Heidrich and Fels, 8), have been prved t be assciated with the presence f the gnadtrpic hrmnes in the urine. These pineer bservatins have been cnfirmed by the large series studies f Zndek (9), Fergusn (0), Herger and Thibaudeau (), Cutler and Owen (). The sex hrmne, estrin,.als seems t be prduced in patients with tumrs and presumably by the tumr tissue itself, as it ccurs in bth male and female subjects. Lewe and his c-wrkers (3) investigated the estrin cn 49

2 50 ALBERT STRICKLER tent f tumr tissue (primary and secndary) remved frm males with extragenital malignant disease and fund this hrmne in abundance. Laqueur and his assciates (quted by Rbsn, 4) bserved a high estrin cntent in the bld f bth male and female patients with malignant neplasms. The significance f the remarkable ccurrence f this hrmne in cnjunctin with rapidly prliferating tissues is nt understd. It is knwn that clstrum cntains a large amunt f estrin and the same hlds true fr cystic cancerus breasts. ANIMAL EXPERIMENTS In the field f animal research there have been a number f cntributins f value. Burrws (5) has shwn that prlnged treatment with estrin may cause desquamative hyperplasia f bth the mammae and the prstate f the muse and that such a cnditin f the breasts is apt t be assciated with cancer in bth man and mice. He finds it difficult, als, in view f these facts, t avid the suspicin that estrin r an altered estrin substance may be respnsible in part fr the prductin f cancer f the prstate. Lacassagne (6) cncludes frm his bservatins that the prlnged actin f estrin n the rganism f the muse manifests itself by the initiatin and maintenance f a multiplying activity f certain tissues, particularly the epithelium f the vagina, uterus, prstate, and breast. But hw can these changes resulting frm cntact with estrin bring abut cancerus transfrmatin? May it nt be pssible that estrin is transfrmed int ne f thse carcingenic hydrcarbns discvered by Kennaway, Ck and their cllabratrs and prved capable f inducing epithelima f the skin after prlnged lcal actin, and sarcma after subcutaneus injectins. These carcingenic agents cntain the same chemical ring which exists in the frmulae f certain rganic substances, as bile salts, chlesterl, and the sex hrmnes. The fact that many f the hydrcarbns pssess an estrgenic as well as a carcingenic prperty cnfirms this affiliatin.. Anther phase f investigatin f interest is the determinatin f the presence f the sex hrmnes in the bld and urine f varius animals. The crpus luteum f the cw is knwn t cntain nly small quantities f estrin. Gnadtrpic hrmnes are excreted in the urine f pregnant mnkeys, but nt f mice, rats, rabbits, dgs, r pigs. The bld f a pregnant mare cntains the gnadtrpic hrmne nly during the first few mnths, while the urine cntains nly Prlan A in small quantities during the early mnths f pregnancy. Lacassagne (7) studied the effect f certain hrmnes, derived frm the urine f pregnant mares, upn mice. Three different substances were islated: estrne, equiline, and equilenine. These substances seem t be endwed with bth estrgenic and carcingenic pwers in varying degrees. Estrne appeared t be the mst ptent carcingenically and equilenine the least. The frmer, when acting in a prlnged and intense manner, was fund capable f prducing characteristic mdificatin f certain rgans, especially the mammary glands, the uterus, the prstate, and hypphysis. In tw strains f mice refractry t cancer, cmbined injectins f estrne and hypphyseal extract caused the prductin f several malignant tumrs.

3 EFFECT OF URINE AND SERA UPON OVARIES 5 TABLE I: Effects f Different Types f Urine and Sera n the Ovaries f Virgin Rabbits Type N. f Animals Ttal Amunt Hemrrhage Cngestin A. Cw serum Steer serum Hg serum Rster serum Chicken serum 5 c.c. 5 c.c, 5 c.c. 5 c.c. 5 c.c. B. Urine frm pregnant wman at ms. Urine frm pregnant wman at 7 ms. 0 c.c. 0 c.c. Urine frm cancer patients Serum btained by injecting urine f cancer patients int bld stream f nrmal chickens, 0-5 c.c, at each dse; ttal c.c, 0 c.c c.c, 3 Serum btained after injecting nrmal chickens with urine f pregnant wmen ( mnths), intravenusly, 0-5 c,c, at each dse; ttal c.c. 5 5 c.c, 3 Serum btained after injecting nrmal chickens with urine f pregnant wmen (7 mnths), intravenusly, 0-5 c.c, at each dse; ttal c.c. (S serum) c.c, 4 Serum made by reinjecting S serum int nrmal chickens 5 c.c. Serum made by injecting S serum int chickens with Rus sarcma 9 5 c.c, 8 Certainly Lacassagne's cnclusins shw a distinct leaning in favr f the carcingenic influence f this hrmne by way f the hypphysis. Anther cmmunicatin f the same general nature is that f Papaniclau (8), in which he reprts his bservatins fllwing the injectin f urine f pregnant cws int immature female guinea-pigs. The varies f the animals which received such injectins did nt shw the frmatin f crpra lutea r the large typical hemrrhagic spts. On the ther hand, there was a definite increase in the vascularizatin f the fllicles and in their atretic disintegratin. Excessive vascularizatin f medium-sized fllicles was ne f the mst utstanding features f the varian changes. Changes were nt limited t the vary and genital tract, but included ther glands, especially the adrenals, the thyrids, and the hypphysis, the adrenals shwing the mst intense effect. Papaniclau inclines twards the belief that the estrgenic hrmne, in additin t its effects n the varian gland, prbably exercises an influence, als, n the vascular system with secndary effects n the varies and with a definite role in the frmatin f the placenta. In the first grup f experiments reprted here the sera f animals used fr fd by man were injected intravenusly int virgin female rabbits and the effect upn the varies was nted. Only 5 c,c. f each serum was injected and the results were read after a twenty-fur hur interval. The

4 5 ALBERT STRICKLER TABLE II: Effect f Pregnant Wman's Urine ( mnths) Frtified with Lecithin and Chlesterl upn the Ovaries f Chickens (U. Urine. L. Lecithin. C. Chlesterl) N. f Animal Ttal Amunt Pathlgical Diagnsis xxx4 50 c.c. U Slight hemrrhage 60 mgm. L 0 mgm, C xxx5 60 e.c, U Slightly hemrrhagic varies 40 mgm. L 60 mgm. C xxx34 80 c.c. U N hemrrhage 360 mgm. L 80 mgm. C xxx 80 c.c, U N hemrrhage 60 mgm. L 0 mgm. C xxx9 70 c.c, U N hemrrhage r bld-pints 80 mgrn. L 96 mgm. C xxxl0 00 c.c, U N hemrrhage 60 mgm. L 8 mgm. C xxxl 90 c.c, U Sme bld-pints 70 mgrn. L 40 mgm. C xxxl l l l 40 c.c, U N hemrrhage 0 mgm. L 0 mgm. C xxx c.c. U Cystic vary 4.5 mgm.l.5 mgm, C xxx c.c. U Cystic vary, marked mgrn, C cngestin and 4 mgm. L hemrrhagic cysts xxx3 40 c.c. U Ovaries nt hemrrhagic 60 mgm, C Beginning vulatin 900 mgm. L xxx39 40 c.c, U Hemrrhagic varies 3000 mgm. L Ovulatin marked 500 mgm, C xxx40 40 c.c, U Prgressive vulatin 3000 mgm, L Bld-pints n varies 500 mgm. C xxx4 40 c,c, U Hemrrhagic varies 3000 mgm, L Ovulatin taking place 500 mgm. C

5 EFFECT OF URINE AND SERA UPON OVARIES 53 TABLE II-Cntinued (U. Urine. L. Lecithin. C. Chlesterl) N. f Animal Ttal Amunt Pathlgical Diagnsis xxx c.c, U Beginning vulatin 5700 rngm. L N hemrrhage 900 rngm, C xxxl c.c, U Ovaries blackened 5400 mgrn. L 800 mgm, C xxx c.c, U Slight vulatin 5400 mgm, L Suggestive hemrrhage 800 mgrn. C xxx75 30 c.c, U Ovulatin ccurring 8000 mgm. L N hemrrhage 600 mgrn, C xxxl76 30 c.c, U Ovulatin ccurring 8000 mgm. L N hemrrhage 600 mgm. C Cystic vary xxx0 300 c.c, U Cngestin, pigmentatin 7500 mgrn, L Cystic vary 500 mgm. C xxx4 0 c,c, U Cystic vary 5500 rngrn. L 00 mgm. C xxx9 00 c.c. U Cystic vary 500 mgm. L 500 mgm. C xxx0 00 c.c, U Cystic vary 500 mgrn, L 500 mgm. C fllwing facts may be gleaned frm Table IA. Nrmal chicken serum was withut effect while nrmal rster serum prduced hemrrhage in the varies in ne instance and cngestin in anther. The serum f a hg prduced nly cngestin; the serum f the steer was withut effect; while the serum f a cw caused hemrrhage in ne instance and cngestin in anther. A study was als undertaken f the effects upn the varies f virgin rabbits, f the serum f Plymuth rck chickens previusly injected with the urine f pregnant wmen at tw mnths and at seven mnths, respectively. These time intervals were selected because during the secnd mnth f pregnancy the excretin f gnadtrpic hrmne is near its peak (S,OO~30,OOO muse units per liter f urine), and at the seventh mnth estrin excretin is at its peak (6,OO~0,OOO muse units per liter f urine). The urine f patients suffering with advanced generalized malignant grwths was als injected int Plymuth Rck chickens and the effect f the serum f these animals upn the varies f virgin rabbits investigated. In these experiments 0 t 5 c.c, f the urine, filtered and f acid reactin, were injected intra-

6 54 ALBERT STRICKLER venusly, daily, until 50 c.c, t 300 c.c. were given. The birds were then sacrificed and the serum btained. Chicken serum s btained was als injected intravenusly int ther chickens suffering frm Rus chicken sarcma and int nrmal chickens, and the effect f sera frm these animals upn the varies f the virgin rabbit was studied. Examinatin f Table IB disclses the fllwing results: The serum f chickens injected with the urine f wmen seven mnths pregnant prduced hemrrhage in the varies f the virgin rabbit in every instance. When this serum was injected int chickens with Rus sarcma and the latter serum was tested as t its effect upn the varies f the virgin rabbit, psitive results (hemrrhage) were btained in eight instances ut f nine. On the ther hand, the serum f chickens treated with the urine f pregnant wmen (tw mnths) and the serum f chickens treated with urine f persns with malignant tumrs did nt yield such cnstantly psitive results. Le Leb (9) injected the urine f pregnant wmen int the peritneal cavity f immature guinea-pigs and fund that this prcedure accelerated the maturatin f the varian fllicles and culd cause vulatin. Urine f pregnant wmen (tw mnths) was injected by us int the peritneal cavity f Plymuth rck chickens. The urine was btained fresh daily, was filtered and f acid reactin. Usually 5 c.c. were injected at ne sitting, althugh in a few instances as much as 50 c.c. were administered. The ttal amunt given varied frm 00 t 500 c.c. The birds were then sacrificed. Autpsy shwed the presence f a certain amunt f urine in the peritneal cavity, at times with a slightly ful dr. There was n evidence f peritnitis. In every case the liver was cvered by a pseud-membrane and shwed grss evidences f degeneratin. The ttal number f chickens s treated was 47, and f this number shwed premature varies; in the varies were nrmal but nn-vulating; and in 4 instances they were in varius stages f vulatin. In 6 instances-ail classed as nn-vulatingthe varies were examined histlgicaily. The micrscpic reprt was cystic varies. The experiments were repeated with varying amunts f lecithin and chlesterl added t the urine befre injectin, these substances being selected because they cnstitute imprtant cnstituents f chicken's eggs. Chlesterl is related t chlalic acid frm which a highly carcingenic agent, methylchlanthrene, has been synthesized (0). Twenty-three chickens were used. The amunt f urine injected daily was the same as in the first experiments but the ttal varied frm as little as 80 c.c. t as much as 380 c.c. The ttal amunts f lecithin and chlesterl varied, respectively, frm 4 mg. and mg. t 5,400 and,800 mg. The latter amunts were the nes ften emplyed. Autpsy shwed absence f peritnitis; little r n urine in the peritneal cavity; pseud-membrane ver the liver slight r entirely absent, and the rgan itself usuaily healthy in appearance. These findings were quite different frm thse in the first grup f experiments. The varies were examined histlgically. As shwn in Table II, in 43 per cent f the animals treated, hemrrhages f varying degree were fund, in 35 per cent vulatin f varying extent was nted, and in 30 per cent the varies were described as cystic.

7 EFFECT OF URINE AND SERA UPON OVARIES 55 SUMMARY. Serum frm chickens injected with the urine f a wman in the seventh mnth f pregnancy, at which time the excretin f estrin is at its height, yielded mre psitive results when injected int virgin female rabbits than the serum f chickens injected with the urine f a wman in the secnd mnth f pregnancy, when the excretin f the gnadtrpic hrmne is at its apex.. Intraperitneal injectin f the urine f a wman in the secnd mnth f pregnancy was withut effect n the varies f a chicken. 3. When lecithin and chlesterl were added t the urine, it prduced hemrrhages in the varies f chickens in 43 per cent f ur series. NOTE: The authr is indebted t Dr. D. derivas, pathlgist, and t Esther Liesner, labratry technician, f the Skin and Cancer Hspital fr their cperatin. BIBLIOGRAPHY. ASCHHEIM, S., AND ZONDEK, B.: Klin. Wchnschr. 6: 3, 97.. SMITH, M. G.: Bull. Jhns Hpkins Hspital 4: 6-66, LAQUEUR, E., DINGEMANSE, E., HART, P. c., AND DEJONGH, S. E.: Klin. Wchnschr. 6: 859, FEE, A. R., MARRIAN, G. F., AND PARKES, A. S.: J. PhysiI. 67: , SOEKEN, G.: Ztschr. f. Kinderh. 53: , OSTERREICHER, W.: Klin. Wchnschr. : 83, ZNDEK, B.: Klin. Wchnschr. 7: 404-4, HEIDRICH, L., FELS, E., AND MATHIAS, E.: "Beitr. z, klin. Chir. 50: , ZONDEK, B.: Klin. Wchnschr. : 74-79, FERGUSON, R. S.: Am. J. Cancer 8: 69-95; HERGER, C. C., AND THIBAUDEAU, A. A.: Am. J. Cancer : , CUTLER, M., AND OWEN, S. E.: Am. J. Cancer 4: 38-35, LOEWE, S., RAUDENBUSCH, W., AND VOSS, H. E.: Bichem. Ztschr. 49: , ROBSON, J. M.: Recent Advances in Sex and Reprductive Physilgy, P. Blakistn's Sn & C., Philadelphia, 934, p BURROWS, H.: Am. J. Cancer 3: 490-5, LACASSAGNE, A.: Am. J. Cancer 7: 7, LACASSAGNE, A.: Am. J. Cancer 8: 735, PAPANICOLAOU, G. N.: Prc. Sc. Exper. BiI. & Med. 8: , LOEB, L.: Prc. Sc. Exper. BiI. & Med. 9: FIESER, L. F., AND NE'YMAN, M. S.: J. Am. Chem. Sc. 57: 96, 935.