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Sodium saccharin, ascorbate and other sodium salts fed at high doses to rats produce urinary bladder urothelial cytotoxicity with consequent regenerative hyperplasia. For sodium salts that have been tested, tumor activity is enhanced when administered either alone or after a brief exposure to a known genotoxic bladder carcinogen. These sodium salts alter urinary composition of rats resulting in formation of an amorphous precipitate. We examined the precipitate to ascertain its composition and further delineate the basis for its formation in rat urine. Using scanning electron microscopy with attached X-ray energy dispersive spectroscopy, the principal elements present were calcium, phosphorus, minor amounts of silicon and sulfur. Smaller elements are not detectable by this method. Infrared analyses demonstrated that calcium phosphate was in the tribasic form and silicon was most likely in the form of silica. Small amounts of saccharin were present in the precipitate from rats fed sodium saccharin (5%), but ascorbate was not detectable in the precipitate from rats fed similar doses of sodium ascorbate. Large amounts of urea and mucopolysaccharide, apparently chondroitin sulfate, were detected in the precipitate by infrared analysis. Chemical analyses confirmed the presence of large amounts of calcium phosphate with variably small amounts of magnesium, possibly present as magnesium ammonium phosphate crystals, present in urine even in controls. Small amounts of protein, including albumin and alpha(2u)-globulin, were also detected (5% of the precipitate). Calcium phosphate is an essential ingredient of the medium for tissue culture of epithelial cells, but when present at high concentrations (5 mM) it precipitates and becomes cytotoxic. The nature of the precipitate reflects the unique composition of rat urine and helps to explain the basis for the species specificity of the cytotoxic and proliferative effects of high doses of these sodium salts.

Dimethylarsinic acid (DMA), fed to rats for 2 years, produced bladder hyperplasia and tumors at doses of 40 and 100 p.p.m., more in females than males. No urothelial proliferation was seen in mice. Our objectives were to investigate the mode of action of bladder tumor formation, evaluate the dose-response and the role of diet and to determine if the urothelial effects were reversible. The study included groups of female F344 rats fed DMA in Purina 5002 diet at doses of 0, 2, 10, 40 or 100 p.p.m. for 10 weeks; two groups of females fed DMA (0 and 100 p.p.m.) in Altromin 1321 for 10 weeks; two groups of males fed DMA (0 and 100 p.p.m.) in Purina 5002 for 10 weeks; a female high-dose recovery group (100 p.p.m. in Purina 5002 diet for 10 weeks followed by control diet for 10 weeks); and two female groups (0 and 100 p.p.m.) in Purina diet for 20 weeks. Urothelial toxicity and hyperplasia were detected by light and scanning electron microscopy (SEM), and the bromodeoxyuridine labeling index was increased in the female 40 and 100 p.p.m. groups. The effects were less in males, but were similar in females fed DMA in Altromin 1321. SEM detected no abnormal urinary solids related to treatment in any group. Urinary calcium was increased in the females fed 40 and 100 p.p.m. in Purina diet, despite overall urinary dilution. Calcification was increased in kidneys of female rats fed Purina diet. The urothelial effects of DMA were reversible. The findings support a non-DNA reactive mechanism for DMA rat bladder carcinogenicity related to urothelial toxicity and regeneration. The toxicity is probably not due to urinary solids. The toxicity and regeneration are produced in a dose-responsive manner in female rats, are greater in female than in male rats, and are reversible.

Uroplakins are the major integral membrane proteins synthesized in terminally differentiated, superficial urothelial cells. Alteration of cell differentiation during rat urinary bladder carcinogenesis was analyzed immunohistochemically for the expression of uroplakins. Expression of uroplakins was compared in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT)-, uracil-, sodium saccharin- or sodium ascorbate-induced urothelial simple hyperplasia, papillary-nodular hyperplasia, papilloma and carcinoma. In controls, uroplakins were located only in superficial cells, especially the luminal surface membrane. In FANFT-induced hyperplasia, including simple hyperplasia, intermediate cells also stained and the staining pattern was disorderly and intermittent. In uracil-induced simple hyperplasia, intermediate cells were stained but in an orderly fashion. In sodium saccharin- or sodium ascorbate-induced simple hyperplasia, superficial cells were swollen but alterations were not observed in the staining pattern. In carcinoma induced by FANFT and uracil, uroplakin expression was very disorderly and focal, usually with no expression on surface cells. It appears that disorderly differentiation is an index of bladder malignancy and is an early event in FANFT-induced lesions but a late event in uracil-, sodium saccharin- and sodium ascorbate-induced lesions.

Sodium saccharin administered at high doses to male rats beginning after 5 weeks of age produces mild urothelial hyperplasia but does not result in a significant increase in incidence of bladder cancer unless it is administered after an initiating agent. However, if it is administered in a two-generation bioassay, a significant incidence of bladder tumors is produced. The hyperplastic and tumorigenic effects are inhibited by co-administration with high doses of NH4Cl. The present experiment was designed to evaluate the effects of another sodium salt, sodium ascorbate, administered through the neonatal time period. Sodium saccharin administered as 5% of the diet produced urothelial hyperplasia and increased labeling index, and this was inhibited by co-administration with 1.23% NH4Cl. Four doses of sodium ascorbate was evaluated. The lowest dose, 0.91%, was without effect on the urinary tract. A slight effect (not statistically significant) was observed at a dose of 2.73%, and a significant proliferative response was detected at 4.56 and 6.84%. Recent studies suggest that a calcium phosphate-containing amorphous precipitate forms in the urine of rats fed high doses of sodium saccharin, producing cytotoxicity of the urothelium and consequent regenerative hyperplasia. This precipitate was observed in the present experiment in the rats administered the high dose of sodium saccharin or the higher doses of sodium ascorbate. Formation of this precipitate and induction of urothelial proliferation were inhibited by co-administration of NH4Cl, but somewhat higher doses of ammonium chloride were required for doses of sodium ascorbate compared to sodium saccharin. These results demonstrate that sodium ascorbate administered through the neonatal time period of the male rat produces urothelial hyperplasia in the dose responsive manner, with a no-effect level of 0.91% of the diet. The formation of the calcium phosphate-containing amorphous precipitate and urothelial proliferation were inhibited by co-administration with NH4Cl.

Propoxur produces bladder tumors in rats, but not other species. The hyperplastic and tumorigenic effects do not occur if urinary pH is lowered by administering propoxur in a semi-synthetic diet or co-administering it with ammonium chloride (NH4Cl). We fed propoxur at 8000 p.p.m. in Altromin 1321 diet to male Wistar rats for 4 weeks, with or without NH4Cl as 10,000 p.p.m. of the diet. The urine of rats fed control diet with or without propoxur had a relatively high urinary pH (approximately 8); the addition of NH4Cl lowered the urinary pH by approximately 0.5-1.0 units. There was no evidence of urinary calculi or amorphous precipitate nor was there an increase in microcrystals or formation of different crystals than occur in normal rat urine. Propoxur produced hyperplasia of the urothelium, as observed by light and scanning electron microscopy, and increased the labeling index for proliferating cell nuclear antigen. These effects were significantly inhibited by co-administration with NH4Cl. There was no evidence of urothelial necrosis. Thus, the hyperplasia appears to result from a direct mitogenic effect of propoxur or a metabolite on the urothelium, rather than from toxicity and consequent regeneration. Based on the present study and previous investigations, the urothelial effects of propoxur in the rat are dependent on high urinary pH and high administered doses, factors which need to be incorporated into any mechanistic model for the chemical and into any extrapolation to potential effects in humans.

N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) administration to rats followed by sodium saccharin results in transitional cell carcinomas of the bladder, of which 24% harbor an activated H-ras gene. Since 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) is the mutagenic and carcinogenic metabolite of FANFT in vivo, we wished to examine ras activation in in vitro ANFT-transformed rat bladder epithelial cells as well as four cell lines established in culture from in vivo FANFT-induced rat bladder tumors. Screening by Western blotting revealed no enhanced levels of p21ras in ANFT-transformed cells nor in cells established in culture from FANFT-induced rat bladder carcinomas. Further investigations using immunohistochemical staining with a different pan-reactive p21 monoclonal antibody (Cetus Corporation) specific for this method, however, showed two groups of cells from FANFT-induced rat bladder tumors had enhanced immunoreactivity. Apart from this, p21ras expression of most of the cells groups varied little from the controls. We examined the reported hot spots (exons 1 and 2) of each of the ras genes (H-, K- and N-ras) by direct sequencing of amplified DNA. No mutations were present. We conclude, therefore, that ANFT transformation of primary rat bladder epithelial cells in vitro may not in this case be mediated by ras activation, although this is difficult to determine since others have observed that optimal culture conditions can select for certain populations of cells without ras activation.

Epidermal growth factor (EGF) and EGF-related growth factors are present in the urine, and EGF has been identified as a urinary component that enhances urinary bladder tumor formation in rats. Neu oncogene encodes a cell surface receptor similar to the EGF receptor and is known to be activated by a point mutation of DNA that encodes the transmembrane domain of the neu protein (p185). In this study, we examined the possible mutational activation of neu oncogene in 50 urinary bladder transitional cell carcinomas (TCC) induced in F344 rats by the following carcinogenesis models: (i) 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (4 weeks)----3% uracil (20 weeks); (ii) 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) (6 weeks)----5% sodium saccharin (72 weeks); and (iii) N-methyl-N-nitrosourea (MNU) 20 mg/kg body wt, i.p. twice per week for 4 weeks----3% uracil (20 weeks). The DNA sequence around the transmembrane domain of neu gene was amplified by PCR and sequenced. The results showed no mutation within the examined DNA sequences, indicating that neu oncogene is not activated by a point mutation in the transmembrane domain in urinary bladder carcinomas induced by BBN, FANFT or MNU.

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. In this study, we investigated the presence of p53 mutations in 122 urinary bladder tumors induced in F344 rats in the following carcinogenesis models: (i) 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT; 6 weeks) in the diet followed by 3% or 5% sodium saccharin in the diet, 5% sodium ascorbate, 3.12% calcium saccharin (CaSac), 1.34% sodium chloride (NaCl), 5.2% CaSac plus 1.34% NaCl, or basal diet alone (72 weeks); and (ii) 0.2% FANFT, 0.05% N-(4-hydroxybutyl)nitrosamine in the drinking water, N-methyl-N-nitrosourea 20 mg/kg body wt, i.p. twice per week, or basal diet alone (4 weeks), followed by 3% uracil in the diet (20 weeks). Polymerase chain reaction-single-strand conformation polymorphism analysis and direct sequencing were performed for exons 5-8 in the rat p53 gene. We found nine tumors (7.4%) with p53 mutations. Two tumors had two mutations in the p53 gene. The tumors that had p53 mutations were relatively smaller than those that did not have p53 mutations. There were no mutation clusters among the treatments or hot-spots for p53 mutations. These results indicate that p53 mutation is infrequent in bladder carcinogenesis in rats, and when it does occur, it does not appear to provide a growth advantage.

Previously, we demonstrated point mutations of the H-ras gene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat urinary bladder carcinomas. In this study, ras oncogene activation was examined in urinary bladder carcinomas induced by N-(4-hydroxybutyl)nitrosamine (BBN) or N-methyl-N-nitrosourea (MNU) administration followed by uracil treatment. In the first experiment, MNU (20 mg/kg body wt) was i.p. injected into 11 male F344 rats twice a week for 4 weeks, followed by feeding 3% uracil for 20 weeks (MNU/uracil group). Ten rats were given only 3% uracil without MNU pretreatment. In the second experiment, 20 male F344 rats were given 0.05% BBN in the drinking water for 4 weeks, then fed 3% uracil for 20 weeks (BBN/uracil group). Another 20 rats were fed 3% uracil without the BBN pretreatment. Transitional cell carcinomas were induced in the urinary bladder of all rats in the MNU/uracil and BBN/uracil groups. Papillomas and hyperplasias were present in the rats given uracil without prior BBN or MNU. DNA and protein were extracted from the tumors (MNU/uracil or BBN/uracil groups) or from the scraped bladder epithelium (uracil alone groups). Sequences around codons 12, 13 and 61 of H-, K- and N-ras genes were examined by direct sequencing after polymerase chain reaction, and p21 was examined by Western blotting. No mutation was found within the examined sequences and p21 showed no changes in mobility. There was no difference in the level of p21 expression between rats treated with MNU/uracil or BBN/uracil compared to corresponding uracil alone groups. These results indicate that the ras oncogene was not activated in urinary bladder carcinomas induced by BBN or MNU in combination with uracil treatment, in contrast to previous findings with FANFT.

High doses of sodium saccharin, a non-genotoxic chemical, lead to the formation of silicate-containing precipitate and microcrystals in urine of male rats. Differences in urinary protein, pH, sodium and other factors affect silicate-containing precipitate and microcrystal formation as well as the bladder effects of sodium saccharin. Total urinary silicon concentration (mostly soluble) in sodium saccharin-fed rats is similar to or lower than the concentration in control rats. Binding of saccharin to male rat urinary proteins was demonstrated by equilibrium-gel filtration. We propose that by binding to urinary proteins under appropriate conditions, saccharin produces a nidus for the formation of silicate-containing precipitate and crystals. These appear to be cytotoxic to the superficial bladder epithelium, with cell death resulting in regenerative hyperplasia. Factors that influence the formation of these silicate-containing materials might provide a rationale for sex, species, dose and dietary differences in response to sodium saccharin.

To establish a rat urinary bladder carcinogenesis model in vitro, primary rat bladder epithelial cells were grown in media containing 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), the water-soluble metabolite of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT), for 4 weeks followed by long-term (4-7 months) exposure to control medium, sodium saccharin (NaS), or urea. Another set of cultures were exposed to ANFT, NaS and urea simultaneously. Several phenotypic changes were observed in the chemically exposed cell cultures, namely differences in cell morphology, increased growth rate and the ability to grow on plastic instead of rat-tail collagen support. All of the chemically exposed cultures were anchorage independent except one of those treated with NaS. The ANFT-treated cells followed by control medium or urea and cells treated with ANFT, NaS and urea were tumorigenic when transplanted to nude mice, whereas NaS or ANFT followed by NaS treatment were not. The tumors were carcinomas and their epithelial differentiation was verified by strong positive staining for cytokeratin. These studies demonstrate the urothelial transforming capability of ANFT in cell culture without the necessity for a long exposure to a secondary chemical.

The mutagenicity of acrolein, allyl alcohol, glycidol and propionaldehyde was measured in V79 cells as resistance to 6-thioguanine. Acrolein was tested with and without fetal bovine serum (FBS) (10%; v/v) during the 2 h incubation period. The concentration of FBS did not affect acrolein toxicity but its mutagenicity declined as the concentration of FBS in the medium rose. Allyl alcohol (AA) was as mutagenic as acrolein (ACR). Glycidol was less mutagenic than AA and ACR. Propionaldehyde was not mutagenic at 1 microM; it was toxic at 2 microM. The data suggest that the mutagenicity of these compounds is mediated by their bifunctional nature whereas their cytotoxicity is mediated by the aldehyde function.

The effect on urothelial proliferation of a urinary bladder carcinogen, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), fed to male F344 rats at doses of 0.2, 0.1, 0.05, 0.01, 0.005 and 0.001% of diet for 4 or 10 weeks was evaluated by autoradiography, using [3H-methyl]thymidine, and by histopathology. At week 4, hyperplasia was induced in 10/11 and 6/11 rats given 0.2% and 0.1% FANFT, respectively. The dose-related increase of labeling index in the bladder epithelium was significant for the groups given 0.01% or higher doses of FANFT. At week 10, histopathologic lesions were observed in groups given 0.05% or higher doses of FANFT. This was accompanied by a significant increase in labeling index for these groups. The results are consistent with the long-term carcinogenicity studies conducted with the same dose levels of FANFT. The interrelationships between numbers of cells (hyperplasia), cell proliferation (labeling index) and cancer induction are discussed utilizing a computerized model of bladder carcinogenesis.

The distribution of intracellular keratin proteins was examined in a variety of urinary bladder lesions of the rat using the immunoperoxidase staining technique. In ethanol-fixed sections, the normal epithelium was strongly positive for keratin staining. Focal regenerative hyperplasia of the bladder epithelium induced by freezing exhibited relatively weak staining. However, diffuse regenerative hyperplasia induced by a single intraperitoneal injection of cyclophosphamide (CP) showed an intensely positive reaction throughout the epithelium. Of the sections fixed with Bouin's solution, the staining reaction was drastically reduced in the normal bladder and the staining was totally negative in the regenerative hyperplasia caused by freezing. Simple hyperplasia induced by a 4-week feeding of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) as 0.2% of the diet exhibited strong reactivity, and nodular and papillarly hyperplasia induced by a 10-week feeding of FANFT was also positive for keratin throughout the lesions. In contrast to the preneoplastic lesions, FANFT-induced transitional cell carcinoma showed differential staining within the tumors. These results suggest that different keratin expression is involved in the proliferative bladder lesions induced mechanically by freezing and chemically by CP or by the carcinogen FANFT.

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) is a potent urinary bladder carcinogen in the rat, and it can be metabolically activated in vitro by a variety of enzyme systems including aerobic cooxidation by prostaglandin H synthase. The latter enzyme is present in the rat bladder mucosa and can be inhibited by the oral administration of aspirin (ASA). To determine if ASA could inhibit the bladder carcinogenicity of FANFT, FANFT (0.2%) was co-administered in the diet with ASA (0.5%) for 12 weeks followed by 1 week of ASA only and then 56 weeks on control diet. 0.2% FANFT followed by control diet induced bladder carcinomas in 18 of 21 (87%) rats, but when ASA was co-administered, only 10 of 27 (37%) rats developed bladder carcinoma (p less than 0.001). However, forestomach tumors, not seen in rats fed only FANFT, developed in 7 rats fed FANFT plus ASA. No tumors occurred in control rats or those fed only ASA. Possible mechanisms, including the role of prostaglandin H synthase in FANFT metabolism, are discussed.

Uracil fed as 3% of the diet to rats produces urinary calculi and consequent proliferative lesions of the bladder epithelium, including papillomatosis. We evaluated the effects of dietary uracil in two strains of mice and compared the results in males and females. Uracil was fed as 3 or 1% of the diet to male and female Swiss and C3H mice for up to 20 weeks. The 3% dose produced marked proliferative changes in the bladder epithelium by 10 weeks of administration, the earliest time at which the animals were processed for microscopic evaluation. These lesions progressed to severe nodular and papillary hyperplasia so that all of the animals fed 3% uracil had to be killed by the end of the 15th week. These animals had uracil-formed calculi. In contrast, mice fed 1% uracil rarely developed uracil calculi, and also rarely developed proliferative changes in the bladder epithelium as observed by light microscopy. Also, the labeling index was determined and showed quantitatively the degree of cell proliferation similar to that qualitatively observed by light microscopic examination. At 10 weeks of administration, there was an increased labeling index in the males compared to females in both strains of mice fed 3% uracil, but this difference was not significant at 15 weeks. Similarly, the males tended to have more severe histologic changes than the females. The results of feeding high doses of uracil are similar in mice to those previously observed in rats.

The co-carcinogenic activity of sodium saccharin and N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was evaluated in male Fischer rats by co-administering them as 5% and 0.005% of the diet, respectively, for 2 years. The effect of simultaneous administration of two urinary bladder promoting substances, sodium saccharin and L-tryptophan as 5% and 2% of the diet, respectively, was also evaluated. Five of 16 rats administered sodium saccharin plus FANFT developed bladder tumors whereas none of the rats administered FANFT, sodium saccharin, or L-tryptophan alone, sodium saccharin plus L-tryptophan, or the control diet developed bladder tumors. Possible mechanisms for the co-carcinogenic activity of sodium saccharin and FANFT are discussed.

A theoretical model of two-stage carcinogenesis has been hypothesized. Variables that are modeled include the populations of normal, initiated, and transformed cells; mitotic rates of these cells; hyperplasia; and the probabilities of cell initiation and transformation during replication. The size of the cell populations can be estimated and mitotic rates determined directly from animal studies. Tumor occurrences at different time intervals following varying periods of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and sodium saccharin administration are known and are used in the indirect estimation of values for unobservable model variables. Model-based analyses suggest FANFT markedly increases the probability of cell initiation in addition to its experimentally verifiable effects on increasing the stem cell population and mitotic rates. Further, experimental results appear inconsistent with the hypothesis that FANFT increases the probability of cell transformation over background levels. Similarly, the effect of sodium saccharin was found to be attributable entirely to increases in stem cell populations and mitotic rates without influencing either the probability of initiation or the probability of transformation.

Uracil has been shown to cause a strong proliferative response in the urinary bladder epithelium of rats and mice through calculus formation and, consequently, acts as a strong promoter in bladder carcinogenesis. In this study, we examined the effect of uracil on two-stage carcinogenesis in various organs using N-methyl-N-nitrosourea (MNU) as the initiator. F344 rats were injected i.p. with MNU twice weekly for 4 weeks and then given diet containing 3.0% uracil for 20 weeks. Uracil induced urinary bladder carcinomas in all rats pretreated with MNU, and it decreased the combined incidence of adenomas and carcinomas in the thyroid. Although not significant, uracil decreased the incidence of adenomas in the lung and increased that of lymphomas of the thymus. A possible influence of a significantly decreased body weight gain caused by uracil treatment on the reduced tumor incidence in the thyroid and lung is discussed. The present data demonstrate the strong promotion activity of uracil for urinary bladder carcinogenesis, and suggest a possible inhibitory effect on thyroid carcinogenesis.