EFFECT OF VARIOUS FACTORS ON INDUCTION OF URINARY BLADDER TUMORS IN ANIMALS BY N-BUTYL-N-(4-HYDROXYBUTYL) NITROSOAMINE

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1 [GANN, 64, ; April, 1973] UDC : [616.62] EFFECT OF VARIOUS FACTORS ON INDUCTION OF URINARY BLADDER TUMORS IN ANIMALS BY N-BUTYL-N-(4-HYDROXYBUTYL) NITROSOAMINE (Plate XXI) Nobuyuki ITO,*1 Kinuko MATAYOSHI,*1 Masayuki ARAI,*1 Yoshio YOSHIOKA,*1 Yoshiyuki KAMAMOTO,*2 Sachio MAKIURA,*2 and Seiichi SUGIHARA*2 (Cancer Center Institute*1 and 1st Department of Pathology,*2 Nara Medical University) Synopsis The effect of various factors on the development of urinary bladder tumor in rats, guinea pigs, and hamsters given N-butyl-N-(4-hydroxybutyl)nitrosoamine was examined histologically. The effect of sex and age, and of the dose and period of administration of N-butyl-N-(4-hydroxybutyl)nitrosoamine on the incidence of urinary bladder tumors in rats was also investigated. No sex difference was found in the incidence of urinary bladder cancer in rats. However, the incidence of cancer and of squamous metaplasia in areas of cancerous tissues was greatly influenced by the age of animals, being much higher in older rats than in younger rats. For development of urinary bladder cancer in rats, the minimum carcinogenic dose of N-butyl-N-(4-hydroxybutyl)nitrosoamine given in the drinking water is 0.005% while 0.001% is the maximum non-carcinogenic dose. On administration of 0.1% carcinogen solution to rats, the minimum period for the induction of urinary bladder cancer is 4 weeks. Guinea pigs and hamsters were less susceptible than Wistar strain rats to the induction of urinary bladder cancer by N-butyl-N-(4-hydroxybutyl)nitrosoamine. INTRODUCTION The carcinogenic activity of N-butyl-N-(4-hydroxybutyl)nitrosoamine was first reported by Druckrey et al.5) Since then, there have been many reports on the histogenesis, histology, and ultrastructure of urinary bladder tumors induced in rats by this carcinogen,13-18,29) and metabolism of this carcinogen21,27) has been studied. Previously, we reported histological and electron microscopic studies on urinary bladder tumors in male Wistar strain rats induced by N-butyl-N-(4-hydroxybutyl)nitrosoamine.15,17) Histologically, these tumors appeared to be transitional cell carcinomas, and squamous metaplasia in areas of transitional cell carcinoma was frequently seen.17) The present paper reports analysis on the effect of various factors on the development of urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosoamine. The factors examined included the sex and age of rats, species difference of animals, and the administration period and concentration of the carcinogen administered. The minimum

2 N. ITO, ET AL. carcinogenic dose and the maximum non-carcinogenic dose of this carcinogen for induction of bladder tumors in rats were also examined. MATERIALS AND METHODS Animals Male and female Wistar strain rats (Fuji Animal Farm, Tokyo) of 4, 8, 12, and 24 weeks old were used. Golden Syrian hamsters and guinea pigs (Fuji Animal Farm. Tokyo) were also used. Rats and hamsters received commercial stock diet MF (Oriental) and guinea pigs received stock diet RC5 (Oriental Yeast Co., Osaka). Carcinogen N-Butyl-N-(4-hydroxybutyl)nitrosoamine (Izumi Chemical Co., Yokohama) was given as 0.1, 0.05, 0.025, 0.01, or 0.001% solution in the drinking water as described previously.17) Experimental Series I A total of 104 male and female rats of 4, 8, 12, and 24 weeks old were given water supplemented with 0.01% N-butyl-N-(4-hydroxybutyl)nitrosoamine for 20 weeks and then water without the carcinogen for 20 weeks. The animals were killed for histological observations 40 weeks after the beginning of the experiment. Experimental Series II Four hundred male rats of 7 to 8 weeks old were given carcinogen solutions of various concentrations for various periods as follows: Group 1: Rats were given water containing 0.1% carcinogen for 2, 4, 6, and 8 weeks and then water without the carcinogen for 38, 36, 34, and 32 weeks, respectively. Control rats received water without the carcinogen for 40 weeks. Group 2: Rats were given water containing 0.05% carcinogen for 2, 4, 6, 8, and 12 weeks and then water without the carcinogen for 38, 36, 34, 32, and 28 weeks, respectively. Group 3: Rats were given water containing 0.01% carcinogen for 2, 4, 6, 8, 12, 20, and 40 weeks and then water without the carcinogen for 38, 36, 34, 32, 28, 20, and 0 weeks, respectively. Group 4: Animals were given water containing 0.005% carcinogen for 2, 4, 6, 8, 12, 20, and 40 weeks and then water without the carcinogen for 38, 36, 34, 32, 28, 20, and 0 weeks, respectively. Group 5: Rats were given water containing 0.001% carcinogen for 40 or 20 weeks. The latter then received water without the carcinogen for 20 weeks. Control rats received water without the carcinogen for 40 weeks. All the animals were killed for histological observation 40 weeks after the start of the experiments. Experimental Series III Twelve male golden Syrian hamsters, 12 guinea pigs, and 12 Wistar strain rats were given water containing 0.025% carcinogen for 20 weeks and were then killed with ether for histological observations. All the animals were housed dying before the time of sacrifice were excluded. The liver and both kidneys were weighed and samples of these organs were taken for histological examination. The urinary bladder was punctured at the urethra, and 0.5ml of 10% buffered formaldehyde solution was injected into the bladder. Then, tissues were fixed and stained with Hematoxylin and Eosin, van Gieson's stain, Mallory's stain, or periodic acid-schiff stain. 152 GANN

3 BLADDER TUMORS BY BBN RESULTS Histological changes in the urinary bladder epithelium were classified into 3 types, hyperplasia (Photo 1), papilloma (Photo 2), and cancer or transitional cell carcinoma (Photo 3), as described previously.17) Areas of squamous metaplasia in the cancer tissue (Photo 4) and invasive growth of cancer cells into the urinary bladder wall were noticed. Cancer in the urinary bladder induced by N-butyl-N-(4-hydroxybutyl)nitrosoamine was usually transitional cell carcinoma. No remarkable changes were seen in the liver, kidney, lung, or gastrointestinal tract. Experimental Series I The average changes in body and liver weights and histological findings in the urinary bladder of the male and female rats in each group are summarized in Tables I and II. All the rats gained weight. Changes were seen in the urinary bladder but not in other organs. Hyperplasia an dpapillomas in the urinary bladder epithelium were seen in rats of both sexes in all age groups. However, the incidence of cancer of the bladder was higher in older rats than in younger ones. The development of squamous metaplasia in cancerous areas also tended to increase with the age of the rats. No sex difference was observed in the incidence of cancer of the bladder, including squamous metaplasia. Table I. Effect of Age on the Incidence of Urinary Bladder Tumors in Male Rats Treated with N-Butyl-N-(4-hydroxybutyl)nitrosoaminea) (BBN) a) 0.01% BBN for 20 weeks and then water for 20 weeks. b) Number of animals (percentage in parentheses). Table II. Effect of Age on the Incidence of Urinary Bladder Tumors in Female Rats Treated with N-Butyl-N-(4-hydroxybutyl)nitrosoaminea) (BBN) a) 0.01% BBN for 20 weeks and then water for 20 weeks. b) Number of animals (percentage in parentheses). 64(2)

4 N. ITO, ET AL. Experimental Series II The average changes in body and liver weight, and histological findings in the urinary bladder of rats which received various concentrations of N-butyl-N-(4-hydroxybutyl)- nitrosoamine in the drinking water for different periods are summarized in Tables III to VII. No cancer of the urinary bladder developed in rats which received 0.001% N- butyl-n-(4-hydroxybutyl)nitrosoamine for 40 weeks. However, it developed in rats Table III. Changes in Body and Liver Weight, and Incidence of Tumors in Male Rats Treated with 0.1% N-Butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for Different Periods * Number of rats (percentage in parentheses) Table IV. Changes in Body and Liver Weight, and Incidence of Tumors in Male Rats Treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for Different Periods * Number of rats (percentage in parentheses) Table V. Changes in Body and Liver Weight, and Incidence of Tumors in Male Rats Treated with 0.01% N-Butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for Different Periods *Number of rats (percentage in parentheses) 154 GANN

5 BLADDER TUMORS BY BBN Table VI. Changes in Body and Liver Weight, and Incidence of Tumors in Male Rats Treated with 0.005% N-Butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for Different Periods * Number of rats (percentage in parentheses) Table VII. Changes in Body and Liver Weight, and Incidence of Tumors in Male Rats Treated with 0.001% N-Butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for Different Periods * Number of rats showing changes Fig. 1. Percentage incidence of focal hyperplasia in the urinary bladder of rats given N-butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) 64(2)

6 N. ITO, ET AL. Fig. 2. Percentage incidence of papilloma in the urinary bladder of rats given N-butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) Fig. 3. Percentage incidence of cancer in the urinary bladder of rats given N-butyl- N-(4-hydroxybutyl)nitrosoamine (BBN) which received 0.005% carcinogen for 40 weeks, 0.01% carcinogen for 6 weeks followed by water without carcinogen for 34 weeks, and in those receiving 0.05% carcinogen for 4 weeks followed by no carcinogen for 36 weeks. On administration of 0.1% carcinogen, no cancer of the bladder was seen in rats which received the carcinogen for 2 weeks and then water for 38 weeks, but 4 of 12 rats (33.3%) developed cancer on receiving this concentration of carcinogen for 4 weeks and then water for 36 weeks. 156 GANN

7 BLADDER TUMORS BY BBN The changes in the bladder (i.e., hyperplasia, papilloma, cancer) on treatment with different doses of the carcinogen for different periods are shown graphically in Figs. 1, 2, and 3. In all the rats in this series, the incidence of hyperplastic lesions of the bladder epithelium was higher than that of papillomas or cancers. The present results show that the incidence of focal hyperplasia, papillomas, and cancers of bladder epithelium increases with increasing concentration of the carcinogen and the period of its administration. The incidence of focal hyperplasia was the highest followed by those of papillomas and cancers, in that order. Experimental Series III The changes in body and liver weight, and histological findings are summarized in Table VIII. Cancers developed only in male Wistar strain rats. No cancer developed in the urinary bladder of guinea pigs or hamsters, though a very low incidence of hyperplasia in the bladder epithelium was observed. Table VIII. Changes in Body and Liver Weight, and in the Urinary Bladder in Rats, Hamsters, and Guinea Pigs Treated with 0.025% N-Butyl-N-(4-hydroxybutyl)nitrosoamine (BBN) for 20 Weeks * Number of animals (percentage in parentheses) DISCUSSION There are many reports on the carcinogenic effect of various chemicals on the urinary bladder of experimental animals.3,4,8,9,11,12,19,20,24,25,28) Some nitroso compounds also have carcinogenic activity on the urinary bladdre,2,22) and especially, N-butyl-N-(4- hydroxybutyl)nitrosoamine has a selective action on the urinary bladder.13,16,17) In general, a sex difference is observed in the incidence of liver cancer induced by various chemical carcinogens.26) However, in the present work, no sex difference was found in the development of urinary bladder cancer in rats. This suggests that the induction of urinary bladder cancer by N-butyl-N-(4-hydroxybutyl)nitrosoamine is not influenced by hormonal factors, but it was found that the development of squamous metaplasia in cancerous lesions was influenced by the age of animals. Metaplasia in human bladder cancer is frequently seen in cancer tissues.8) These findings suggest that the development of squamous metaplasia may be influenced by age. In this work, the minimum carcinogenic dose of N-butyl-N-(4-hydroxybutyl) nitrosoamine for induction of urinary bladder cancer in rats and the maximum non-carcinogenic dose were investigated. Recently, it has been suggested that focal hyperplasia of the bladder epithelium is a pre-cancerous lesion.6,7,16) In support of this, administration of various doses of carcinogen for various periods in the present work showed that the incidence of focal hyperplasia was consistently higher than that of papillomas or cancers, and hyperplasias de- 64(2)

8 N. ITO, ET AL. veloped before other lesions of the bladder. These findings on hyperplastic foci confirm our previous findings on the carcinogenic effect of N-butyl-N-(4-hydroxybutyl)nitrosoamine on the urinary bladder in rats.13,16) There have been a few reports on the carcinogenic activity of this compound on the urinary bladder of mice and dogs.1,23) From these reports the incidence and development of cancer and the histological patterns observed in these animals seem to be essentially similar to those seen in rats. However, in the present work, hamsters and guinea pigs were much less sensitive than rats to the induction of bladder cancer by N-butyl- N-(4-hydroxybutyl) nitrosoamine. Okada et al.21) reported the metabolism of N-butyl-N-(4-hydroxybutyl)nitrosoamine, and they observed that on treatment of rats with N-butyl-N-(4-hydroxybutyl)nitrosoamine, the carcinogen was not recovered in the urine, but that about 20% of the dose was recoverd as butyl(3-carboxypropyl)nitrosoamine in the urine. After oral administration of butyl(3-carboxypropyl)nitrosoamine to rats about 45% was recovered unchanged in the urine. The carcinogenic activity of butyl(3-carboxypropyl)nitrosoamine on the urinary bladder of rats was reported by Hashimoto et al.10) Recently, Suzuki et al.27) reported that the urinary content of butyl(3-carboxypropyl)nitrosoamine in guinea pigs treated with N-butyl-N-(4-hydroxybutyl)nitrosoamine was very low. These findings suggest that the urinary concentration of the metabolite, butyl (3-carboxypropyl)- nitrosoamine, may be important in inducing bladder cancer in rats. From the present work, it seems that a difference in the sensitivity of different species of animals to the carcinogen may be due to the difference in the concentration of butyl(3-carboxypropyl)- nitrosoamine in the urine. Even in hamsters and guinea pigs a very low incidence of focal hyperplasia of the bladder was observed on treatment with N-butyl-N-(4-hydroxybutyl)nitrosoamine. Thus, if the period of observation were longer or the dose of carcinogen were higher than those in the present work, it might be possible to induce bladder cancer in hamsters and guinea pigs with N-butyl-N-(4-hydroxybutyl)nitrosoamine. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, by a grant from the Tokyo Biochemical Research Foundation, and by a grant from the Experimental Pathological Research Association. (Received November 24, 1972) REFERENCES 1) Akagi, G., Akagi, A., Kimura, M., Proc. Japan. Cancer Assoc., 29th Annu. Meet., 65 (1970). 2) Bertram, J. S., Craig, A. W., Brit. J. Cancer, 24, 352 (1970). 3) Bonser, G. M., Clayson, D. B., ibid., 36, 26 (1964). 4) Clayson, D. B., Cooper, E. H., Adv. Cancer Res., 13, 271 (1970). 5) Druckrey, H., Preussmann, R., Ivankovic, C., Schmidt, C. H., Mennel, H. D., Stabl, K. W., Z. Krebsforsch., 66, 280 (1964). 6) Erturk, E., Cohen, S. M., Prise, J. M., Bryan, G. T., Cancer Res., 29, 2219 (1969). 7) Erturk, E., Price, J. M., Mcris, J. E., Cohen, S., Leith, R. S., von Esch, A. M., Croretti, A. J., ibid., 27, 1988 (1967). 8) Friedman, N. B, Ash, J. E., "Tumors of the Urinary Bladder", (1959). Armed Forces Institute of Pathology, Washington, D.C. 9) Ghindoni, J. J., Campbell, M. M., J. Pathol., 67, 665 (1969). 158 GANN

9 BLADDER TUMORS BY BBN 10) Hashimoto, Y., Noda, M., Suzuki, K., Kurashima, Y., Okada, M., Proc. Japan. Cancer Assoc, 31st Annu. Meed., 9 (1972). 11) Hicks, R. M., Chem.-Biol. Interactions, 1, 49 (1969). 12) Hicks, R. M., Wakefield, J. St. J., ibid., 5, 139 (1972). 13) Ito, N., Acta Pathol. Japon., 22, (1973), in press. 14) Ito, N., Hiasa, Y., Kamamoto, Y., Makiura, S., Sugihara, S., Marugami, M., Okajima, E., GANN, 62, 435 (1971). 15) Ito, N., Kamamoto, Y., Makiura, S., Sugihara, S., Arai, M., Matayoshi, K., Sugihara, R., Kitamura, H., Proc. Japan. Cancer Assoc., 31st Annu. Meet., 269 (1972). 16) Ito, N., Hiasa, Y., Kamamoto, Y., Makiura, S., Yokota, Y., Matayoshi, K., Okajima, E., II. Int. Symp. Princess Takamatsu Cancer Res. Fund., 11 (1971). 17) Ito, N., Hiasa, Y., Tamai, A., Okajima, E., Kitamura, H., GANN, 60, 401 (1969). 18) Ito, N., Makiura, S., Yokota, Y., Kamamoto, Y., Hiasa, Y., Sugihara, S., ibid., 62, 359 (1971). 19) Koss, L. G., Lavin, P., J. Natl. Cancer Inst., 46, 585 (1971). 20) Lavin, P., Koss, L. G., ibid., 46, 597 (1971). 21) Okada, M., Suzuki, E., GANN, 63, 391 (1972). 22) Okajima, E., Hiramatsu, T., Motomiya, Y., Iriya, K., Ijuin, M., Ito, N., ibid., 62, 163 (1971). 23) Okajima, E., Hiramatsu, T., Motomiya, Y., Iriya, K., Ijuin, M., Kondo, T., Hirao, Y., Matsushima, S., Proc. Japan. Cancer Assoc. 31st Annu. Meet., 10 (1972). 24) Oyasu, R., Battifora, H. A., Aisenstein, R., McDonald, J. H., Hass, G. M., J. Natl. Cancer Inst., 40, 377 (1968). 25) Oyasu, R., Miller, D. A., McDonald, J. H., Hass, G. M., Arch. Pathol., 75, 184 (1963). EXPLANATION OF PLATE XXI Photo 1. Area of focal hyperplasia in the bladder epithelium of a rat treated with N-butyl-N- Photo 2. Papilloma of the bladder epithelium in a rat treated with N-butyl-N-(4-hydroxybutyl)- Photo 3. Transitional cell carcinoma of the bladder in a rat treated with N-butyl-N-(4-hy- Photo 4. Squamous metaplasia in an area of transitional cell carcinoma in the bladder of a rat, H-E=Hematoxylin and Eosin stain 64(2)

10 GANN, Vol. 64 PLATE XXI