The Role of the Urine in Vesical Neoplasm. 1. Experimental Confirmation of the Urogenous Theory of Pathogenesis

THE JOURNAL OF UROLOGY

Vol. 71, No. 5, May 1954 Printed in U.S.A.

THE ROLE OF THE URINE IN VESICAL NEOPLASM. 1. EXPERIMENTAL CONFIRMATION OF THE UROGENOUS THEORY OF PATHOGENESIS DONALD F. McDONALD

AND

ROLAND R. LUND

From the Division of Genito-Urinary Surgery of the University of Washington School of Medicine, Seattle, Wash.

The problems prompting this experiment were twofold. Do the urinary constituents play an important part in producing experimental bladder tumors? Secondly, will established experimental tumors regress when deprived of contact with urine? Both questions have been answered. The carcinogen reached the bladder in effective concentration only in the urine. Once established the tumors no longer required continued contact with carcinogen or urine. METHODS

In ten young adult female mongrel dogs and twenty-nine adult male rabbits the dome half of the urinary bladder was surgically transformed into a pouch isolated from contact with the urine. The method varied slightly in the two species but in all cases care was taken to preserve the blood supply to the pouch (fig. 1). The choice of sex in the two species was determined by the greater ease of cystoscopy in female dogs and male rabbits. At the time of surgery the bladders were inspected to rule out pre-existing lesions. The animals received the carcinogen, beta-naphthylamine orally. Capsules containing 600 mg. were given daily to the dogs. A 0.1 per cent solution of betanaphthylamine hydrochloride was given ad libitum to the rabbits in place of drinking water. The daily voluntary intake averaged 350 mg. The hydrochloride of beta-naphthylamine was prepared freshly because it deteriorated. The animals were weighed weekly. The diet of the rabbits consisted of commercial pellets, fresh lettuce and carrots. The dogs received canned dog food and meal and fresh horse meat. Cystoscopy was performed at monthly intervals with the 16F cystoscope. This was accomplished without the need for anesthesia in rabbits but dogs required general anesthesia. When well-established tumors were demonstrated, the dogs were explored surgically, the lesions photographed and biopsied, and the urinary stream diverted. Ureterovaginostomy and transplantation of the ureters to the pouch with drainage of the pouch by polyethylene cystostomy tube were more successful methods than ureterosigmoidostomy. Following deviation of the urinary stream no further carcinogen was administered. The method of Bratton and Marshall was used for the determination of urinary aromatic amine. This work was supported in part by a Grant-in-aid from the National Cancer Institute of the U.S. Public Health Service, by Initiative 171 Research Funds provided by the State of Washington for research in the biological and medical sciences and by the John and Mary R. Markle Foundation. Accepted for publication May 4, 1953.

560

ROLE OF URINE IN VESICAL NEOPLASM

561

FIG. 1. Method of preparing bladder pouch in dogs. From left to right: incision in antenor wall of bladder, mobilization and incision of mucosa, suture of mucosa and muscularis separately, external appearance of completed pouch.

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2. 1-at wa//-"30 "/4 "5.Neck- O %

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5. Vault ordome-Z7.5% 6.Antwall -5 %

FIG. 2. Location of first tumors seen on cystoscopy in dogs treated with beta-naphthylamine. RESULTS

1) Dogs. As early as two weeks after initiating treatment intermittent melena (four plus benzidine reaction) occurred in five animals. This was more prevalent with beta-naphthylamine than with the hydrochloride. Gastro-intestinal bleeding was never a serious problem. By two months all dogs showed microscopic hematuria which continued throughout the experiment. The first cystoscopic lesion seen was hyperemia about the ureteral orifices after an average latent period of nine months. After an average additional interval of two months, telangiectases were noted in the bladder wall. Over some of these telangiectases small brown spots suggestive of epithelial thickening were noted. The thickened epithelium in some cases resembled cystic bullous lesions. The dark brown spots and the cystic lesions were observed to enlarge until recognizable as papillary tumors. These were delicate and bled easily when traumatized. Some of the tumors grew to large sessile infiltrating carcinomas. The locations for the first tumors were: posterior wall 33.5 per cent, lateral walls 30 per cent, dome 27.5 per cent, trigone 6 per cent, anterior wall 3 per cent (fig. 2). After the first tumor appeared others developed with remarkable rapidity. As many as eight new tumors appeared in one month. Four dogs eventually developed papillomatosis covering the entire interior surface of the urinary bladder. In one animal the neoplasm extended into the urethra. Table l summarizes the relationship of total dose to latent period and position of the first tumors seen. Tumors were absent from the pouches of all dogs except one in

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DONALD F. McDONALD AND RONALD R. LUND

TABLE

DOG

1. Relationship between ti,mor induction time, total dose of carcinogen and number and location of first observed tumors /3-NAPHTHYLAMINE TOTAL DOSE, GRAMS

54

136

37 39 41 35

124 124 139 150

34

INDUCTION PERIOD, MONTHS

NUMBER OF TUMORS

874

3

11 11 11 12,%

1 1 2 6

234

13½

3

36 32

247 280

13½ 13%

2 7

45

590

16

3

33

258

17

4

I

LOCATION OF TUMORS

1 posterior wall 1 right posterior wall 1 left posterior wall Right lateral wall Right lateral wall Posterior wall 3 dome 3 left posterior wall 2 dome 1 right trigone 2 left ureteral orifice 3 posterior wall 4 right posterior wall 1 right anterior wall 2 posterior wall 1 right lateral wall 3 dome

Fm. 3. Dog 34 bladder photograph looking down at vesical neck showing multiple small

tumors.

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FIG. 4. Dog 34 pouch. Multiple tumors occurring in pouch due to communication (rubber tubing inserted in ostium for demonstration purposes) with bladder which permitted contact of urine with epithelium of pouch. This was the only pouch which was found to contain tumors.

which there was a communication of the pouch with the bladder which permitted a free flow of urine into the pouch (figs. 3 and 4). In no case did tumors develop in bladder epithelium not in contact with the urine. The excretion of aromatic amines in the urine of the treated dogs was found to vary from 1.25 to 4.5 mg. per ml. with an average of 3.3 mg. per ml. in 33 tests. Normal dogs excrete an average of 1.0 mg. per ml. of aromatic amine. Fluid aspirated from bladder pouches did not give the qualitative test for aromatic amines. Histological grading of the tumors by the method of Col. J. E. Ash of the Army Institute of Pathology showed variation from grade one to three transitional cell carcinoma with squamous cell components in eight specimens (fig. 5). Most of the tumors were papillary and of grade two malignancy. Extensive invasion of the bladder wall was noted in six. No distant metastases were found. In one section a tumor embolus in a blood vessel was noted (fig. 6). Nearly all of the tumors showed adjacent submucosal leukocytic infiltration. Some showed frank necrosis and infection. Hyperemia was always present at the base of the tumor. The cell nests of von Brunn, which are submucosal groups of urinary epithelial cells, were observed to be hyperplastic in half of the specimens. In one dog these nests developed into cystitis cystica and cystitis glandularis. The cell nests had no constant relationship to the tumors.

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DONALD F. McDONALD AND RONALD R. LUND

FIG. 5. Dog 32. Sessile papillary carcinoma after sixteen months' treatment with betanaphthylamine.

FIG. 6. Dog 32. Grade III transitional cell carcinoma with epithelial pearl formation. Note tumor embolus in blood vessel at right lower margin of cut.

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Frn. 7. Dog 36. Bladder at time of exploration and just prior to deviation of urine. Multiple small tumors are present.

The urine was successfully deviated from the tumors in five dogs. After one, two, three, four and one-half and five months of freedom from contact with the urine the neoplasms continued to grow (figs. 7, 8, 9 and 10). There was neither gross nor microscopic evidence of r~gression. No papillary tumors were found in the ureters or renal pelves. In two dogs hyperplasia and renal tubular adenoma formation were seen (fig. 11). In one section an early transitional cell carcinoma was seen in the renal medulla (fig. 12). Toxic effects were produced by beta-naphthylamine. One dog died after six months of treatment, probably due to distemper. She received 200 mg. daily of the hydrochloride for a total dose of 36 gm. Death occurred one week after hematuria became marked. Necropsy showed hemosiderin deposits and vacuolization of the renal tubules. There were granular and red cell casts in the collecting tubules. Routine post-mortem examinations of other dogs treated for much longer periods showed extensive hemosiderosis of the kidney, liver and spleen. Slight renal tubular degeneration and fatty infiltration of the liver were common. Chronic purulent vaginitis and pyelonephritis occurred in all dogs subject to ureterovaginostomy. Areas of hyperemia in the intestinal mucosa were seen in two animals but no other lesions of the gastro-intestinal tract were observed. All dogs gradually lost weight during the two year study. This, however, was not of such degree that it interfered ·with the general health and activity of the animal with one exception. A Collie required several two to three week interruptions of treatment to restore its appetite and activity. 2) Rabbits. No neoplastic lesions were noted in the bladder or pouch of rabbits after two years of treatment. Other proliferative lesions occurred as shown in table 2. Proliferation of von Brunn's cell nests was noted in the area of the healing mucosa within one month after the first operation. Two other rabbits showed

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DONALD F. McDONALD AND RONALD R. LUND

FIG. 8. Dog 36. Bladder three months after deviation of the urinary stream and discontinuance of beta-naphthylamine. Multiple large tumors are present.

Fm. 9. Dog 41. Biopsy of tumor taken at time of deviation of urine. Note lack of invas10n.

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Fm. 10. Dog 41. Highly invasive carcinoma of bladder 1 month after deviation of the urine

Fm. 11. Dog 34. Tubular adenomas and transitional cell carcinoma of kidney after 16 months' exposure to beta-naphthylamine.

these changes in both the bladder and pouch after three months and nine months. Twelve rabbits showed cystitis cystica and cystitis glandularis after an average induction period of 21 months with a range of twelve to twenty-four months. Grossly, the lesions varied from minute solitary structures to a thickened mucosa

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DONALD F. McDONALD AND RONALD R. LUND

Fm. 12. Dog 34. Early transitional cell carcinoma of renal pyramid TABLE

2. Proliferative lesions in rabbits treated with beta-naphthylamine LESION

Von Brunn's cell nests. Cystitis cystic a and cystitis glandularis. Bladder polyps . Squamous metaplasia of bladder. Epithelial hyperplasia of renal pelvis. Leucemia. . ....... . Proliferation of bile ducts.

NUMBER OF ANIMALS

3 12 2

3

7 1 2

TREATMENT TIME

4 21 20 23 24 9 24

months months months months months months months

resembling tapioca on surface view and resembling sponge rubber on cut section. In five of these animals the cystitis was found in both the bladder and the pouch. In six the cystitis was found in the bladder only; and in one, the pouch only. Those pouches having the lesions were infected. Microscopically the lesions were typical. Moderate round cell infiltration was observed in the region of the cysts. Two rabbits developed polyps consisting largely of connective tissue with some infiltration by plasma cells and lymphocytes. There was no change in the epithelium which was focally absent. Squamous metaplasia of the bladder was found in three rabbits and hyperplasia of the renal pelvis epithelium was found in seven rabbits at twenty-four months. Lesions of a neoplastic nature occurred in three rabbits. One of these developed leucemia after nine months. The lungs, liver, kidneys and most other organs of the body were infiltrated with the leucemia cells. Two other animals, sacrificed after two years were found to have multiple heptic nodules which microscopically were adenomatous proliferations of bile ducts with well differentiated structure. In one rabbit ectopic bone formed in the submucosa of the pouch.

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Cyanosis appeared in rabbits given 0.2 per cent beta-naphthylamine hydrochloride solutions ad libitum in lieu of drinking water after three weeks exposure. Cyanosis was marked in seven rabbits and present in five others. Reduction of the concentration to 0.1 per cent prevented cyanosis. Hematuria did not occur in rabbits. Post-mortem examination showed a urinary tract infection with pyelonephritis in six animals and chronic inflammatory changes in the bladder of most animals. Renal tubular degeneration and hemosiderosis in the liver, spleen and kidney were characteristic findings. Rabbits who excreted primary amines in acetylated form were found to have no free aromatic amines in the urine as opposed to the dogs who excreted it all in free form. After hydrolysis there was range of 1.15 to 5.5 mg. per ml. with an average of 3.0 mg. per ml. in 19 determinations on rabbit urine. DISCUSSION

The experiments clearly show that urinary constituents have an etiologic role in the production of beta-naphthylamine bladder tumors in dogs. In no case did carcinoma develop in epithelium not in contact with urine. In every dog carcinoma developed in the bladder epithelium in contact with the urine. If a blood-borne carcinogen were responsible for tumor growth, then neoplastic change should have developed in the bladder epithelium in the pouch which had an intact blood supply. Neoplastic change occurring in the pouch of the animal whose pouch communicated with the bladder and hence contained urine demonstrated that the pouch epithelium was just as susceptible as the bladder epithelium when properly stimulated. The active urinary carcinogen was not identified in this study but the work of Mellors and Hlinka indicates that it is present in both blood and urine. Bonser, Clayson and Jull showed that 2-amino-1-naphthol, the probable active metabolite of beta-naphthylamine, is 200 times more concentrated in the urine than in the blood of dogs. These observations in addition to the direct evidence of the present experiment substantiate the urogenous theory of vesical carcinogenesis. Even though the active carcinogen is present in both the blood and the urine it is effective only in the high concentration found in the urine. The fact that hyperemia and telangiectasis were the first lesions noted on cystoscopy does not necessarily indicate a hematogenous route as Ferguson and Gay advocate. Rather it may indicate that the urine-borne carcinogen is acting as an irritant with resulting hyperemia, and/ or that the growth stimulating properties of the carcinogen have progressed to the point of preparing a blood supply for the newly forming epithelial tumor. The primary location of the tumors does not correspond with the findings of Hueper, ·Wiley and Wolfe who found induced tumors to be mostly on the dependent portions of the bladder. In this study very few tumors appeared on the anterior wall. It is certainly true that all surfaces of the urinary bladder are constantly wet with urine and dependency can play only a minor role. In the rabbits the cystitis cystica and glandularis, the polyps and squamous metaplasia are interpreted to be nonspecific lesions due to trauma and infection. Hueper's observation (Hue per, Briggs and vVolfe) that in experimental bladder

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DONALD F. McDONALD AND RONALD R. LUND

tumors distant metastases are rare but local invasion is common is confirmed. This is also true in humans (Stirling and Ash; Waller and Hamer). All of the dogs developed tumors despite a mixed genetic background although it is true that there was a variation in the quantity of carcinogen apparently required and the time of induction. The intensity of the stimulus in our experiment, 600 mg. daily, as compared with 450 mg. given in Hueper's series, reduced the average induction time from twenty months to twelve and three-fourths months. Deviation of the urine and cessation of the carcinogenic stimulus did not inhibit the growth of the tumors. This is the usual observation in cancer. Once the process is firmly established it continues without further need of exogenous stimulation. Regression of bladder tumors in humans has been reported following deviation of the urine (Davis; Abeshouse and Scherlis) but often no beneficial influence derives from this procedure. We were not able to confirm by this experiment that deviation of the" urine from established tumors results in their regression. The renal adenoma and early transitional cell carcinoma of the duct of Bellini are interpreted as neoplastic responses to the beta-naphthylamine. To our knowledge such lesions have not been previously reported. The leucemia in one rabbit may have been coincidental although spontaneous leucemia in rabbits is rare. The hepatic duct proliferations in two rabbits may have also been coincidental. CONCLUSIONS

Bladder tumors can be easily induced in dogs by beta-naphthylamine. Rabbits are resistant to induction of beta-naphthylamine tumors in the dose and time selected, but hyperplasia and metaplasia may result. Epithelial contact with the urine is essential to the production of experimental bladder tumors in dogs. This confirms the urogenous theory of vesical carcinogenesis. Deviation of the urine from established experimental bladder tumors does not inhibit their growth. Renal adenoma and early transitional cell carcinoma of the collecting duct epithelium may be induced by beta-naphthylamine.

B-511 Health Sciences Bldg., University of Washington, Seattle, Wash. REFERENCES ABESHOUSE, B. S. AND ScmrnLis, I.: Urol. and Cut. Rev., 55: 1, 1951. AsH, J.E.: J. Urol., 44: 135, 1940. BONSER, G., CLAYSON, D. AND JuLL, J.: Lancet, 261: 286, 1951. BRATTON, A. C. AND MARSHALL, E. K., JR.: J. Biol. Chem., 128: 537, 1938. DAVIS, E.: J. A. M.A., 137: 450, 1948. FERGUSON, R. S.: J. Urol., 31: 122, 1934. GAY, D. M.: J. Urol., 31: 126, 1934. HUEPER, W. 0., WILEY, S. H. AND WoLFE, H. D.: J. Ind. Hyg. and Toxicol., 20: 46, 1938. HuEPER, W. 0., BRIGGS, F. R. AND WoLFE, H. D.: .J. Ind. Hyg. and Toxicol., 20: 85, 1938. MELLORS, R. AND HLINKA, J.: Cancer, 5: 242, 1952. STIRLING, C. AND AsH, J.E.: J. Urol., 45: 342, 1941. WALLER, J. I. AND HAMER, H. G.: J. Urol., 64: 651, 1950