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Comparative Bladder Tumor Promoting Activity of Sodium Saccharin, Sodium Ascorbate, Related Acids, and Calcium Salts in Rats¹

Department of Pathology and Microbiology [S. M. C., L. B. E., T. O., T. M., S. L. J., R. A. S., J. M. W., M. K., E. M. G.] and the Eppley Institute [S. M. C., S. L. J.], University of Nebraska Medical Center, Omaha, Nebraska 68198-3135; Cantox, Inc., Oakville, Ontario, Canada, L6J 6J5 [C. I. C.]; Charlottesville, Virginia 22901 [G. P. S.]; and International Life Sciences Institute/Nutrition Foundation, Washington, D.C. 20036 [J. L. E.]

Sodium saccharin and sodium ascorbate are known to promote urinary bladder carcinogenesis in rats following initiation with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-butyl-N-(4-hydroxybutyl) nitrosamine. Sodium salts of other organic acids have also been shown to be bladder tumor promoters. In addition, these substances increase urothelial proliferation in short term assays in rats when fed at high doses. When they have been tested, the acid forms of these salts are without either promoting or cell proliferative inducing activity. The following experiment was designed to compare the tumor promoting activity of various forms of saccharin and to evaluate the role in promotion of urinary sodium, calcium, and pH as well as other factors.

Twenty groups of 40 male F344 rats, 5 weeks of age, were fed either FANFT or control diet during a 6-week initiation phase followed by feeding of a test compound for 72 weeks in the second phase. The chemicals were administered to the first 18 groups in Agway Prolab 3200 diet and the last 2 groups were fed NIH-07 diet. The treatments were as follows: (a) FANFT 5% sodium saccharin (NaS); (b) FANFT 3% NaS; (c) FANFT 5.2% calcium saccharin (CaS); (d) FANFT 3.12% CaS; (e) FANFT 4.21% acid saccharin (S); (f) FANFT 2.53% S; (g) FANFT 5% sodium ascorbate; (h) FANFT 4.44% ascorbic acid; (i) FANFT 5% NaS plus 1.15% CaCO₃; (j) FANFT 5.2% CaS plus 1.34% NaCl; (k) FANFT 5% NaS plus 1.23% NH₄Cl; (l) FANFT 1.15% CaCO₃; (m) FANFT 1.34% NaCl; (n) FANFT control; (o) control 5% NaS; (p) control 5.2% CaS; (q) control 4.21% S; (r) Control control; (s) FANFT 5% NaS (NIH-07 diet); (t) FANFT control (NIH-07 diet).

NaS, CaS and S without prior FANFT administration were without tumorigenic activity.

NaS was found to have tumor promoting activity, showing a positive response at the 5 and 3% dose levels, with significantly greater activity at the higher dose. CaS had slight tumor promoting activity but without a dose response, and S showed no tumor promoting activity. In addition, NaCl showed weak tumor promoting activity, but CaCO₃ was without activity. NH₄Cl completely inhibited the tumor promoting activity of NaS when concurrently administered with it. NaCl administered with CaS or CaCO₃ administered with NaS showed activity similar to that of NaS. Sodium ascorbate was also shown to have tumor promoting activity, with slightly less activity than NaS. Ascorbic acid showed no tumor promoting activity. NaS when administered in Prolab diet had greater tumor promoting activity than when administered in NIH-07 diet.

FANFT administration produced an increase in urinary pH, which remained elevated compared to control fed rats even after the FANFT was discontinued from the diet. NaS treated rats had a higher urinary pH than rats fed CaS, and S produced an even lower urinary pH. Rats fed NaS, CaS, or S after FANFT had higher urinary pH values than rats fed these chemicals after control diet. When administered with NaS, NH₄Cl markedly acidified the urine, and CaCO₃ raised the urinary pH in comparison to the urinary pH of rats fed NaS alone. The urinary pH of rats fed NIH-07 diet was consistently lower than comparable groups fed Prolab 3200 diet.

The results of these experiments strongly suggest that enhancing factors for the tumorigenicity of bladder tumor promoters such as NaS and sodium ascorbate are increased urinary sodium concentration and urinary pH greater than approximately 6.5. Calcium does not appear to enhance or inhibit the tumor promoting activity of these compounds directly but might have effects due to acidification of the urine if administered as a salt such as calcium saccharin. Also, some evidence is presented that urinary volume may be a contributing factor to the tumor promoting activity of these compounds.

¹ Supported by USPHS Grants CA32513 and CA36727 from the National Cancer Institute and by a grant from the International Life Sciences Institute-Nutrition Foundation.

² Havlik-Wall Professor of Oncology. To whom requests for reprints should be addressed, at Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-3135.

Received 10/ 8/90. Accepted 1/21/91.

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