Comparison of the responses of male rats to dietary sodium saccharin exposure initiated during nursing with responses to exposure initiated at weaning

In an attempt to define the role of exposure to sodium saccharin (NaS) during early life on the subsequent development of bladder tumours, we compared the responses of male rat pups to exposure to 5% dietary NaS initiated at parturition with those to exposure initiated at weaning. We also compared the effects of exposure from parturition to NaS given in a low-carbohydrate (L-CHO) diet with those of NaS in rat chow. NaS ingestion by the dam was associated with low saccharin concentrations in the pups' urine and had no effect on the caecal or bladder mass in the suckling pups. In the 10 wk after weaning, the rats ingesting NaS in chow showed decreased weight gain and increases in feed consumption, mass of caecal contents and tissue, urine output, bladder mass, relative water consumption (g water consumed/g feed consumed) and bladder hyperplasia. Except for bladder hyperplasia these effects were generally greater in the rats exposed to NaS from parturition than in those exposed only from weaning. The animals exposed to NaS in the L-CHO diet had the highest level of urinary saccharin but showed no bladder hyperplasia. The significance of these findings to the role of pre-weaning saccharin exposure in bladder tumorigenesis is discussed, and it is concluded that the effects on urinary parameters and the bladders of rats exposed to NaS during suckling and weaning may be secondary to the effects of NaS on the gastro-intestinal tract.     

The effect of long-term administration of aspirin and sodium saccharin on the rat kidney

In a study primarily designed to evaluate the inhibitory effects of aspirin on N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-initiated and saccharin-promoted bladder carcinogenesis, significant renal lesions were observed. Thus, administration in the diet of aspirin and sodium saccharin to F344 male rats for 68 weeks resulted in significant lesions of the renal papilla. In contrast to the bladder, aspirin enhanced the frequency and severity of the proliferative action of sodium saccharin on the epithelium of the renal papilla (p less than 0.05 compared to rats treated with either compound alone). The majority of rats administered the two chemicals together demonstrated moderate to severe urothelial hyperplasia of the renal papilla. Columnar metaplasia of the papillary epithelium also occurred frequently in rats fed the combination of chemicals. The rats treated with a combination of sodium saccharin and aspirin had a high incidence of renal papillary necrosis which was also present to a lesser extent among rats treated with aspirin only. Papillary calcification was also frequently observed in the rats fed the combination of aspirin and sodium saccharin. Sodium saccharin or aspirin alone reduced the light microscopic incidence and severity of rat nephropathy, a common finding in aging rats. It would appear that the hyperplastic and renal papillary toxic effects of aspirin and sodium saccharin are independent responses, and that the administration of the two chemicals together greatly accentuates these responses.     

Promoting effects of various chemicals in rat urinary bladder carcinogenesis initiated by N-nitroso-n-butyl-(4-hydroxybutyl)amine

We studied the capacity of various chemicals to promote urinary bladder cancer in male F344 rats after initiation by N-nitroso-n-butyl-(4-hydroxybutyl)amine (BBN). The rats were given initially 0.01% BBN in the drinking-water for 4 wk and then the test compound in the diet for 34 wk. Effects were judged by measuring the formation of preneoplastic lesions papillary or nodular hyperplasia (PN hyperplasia) of the urinary bladder. Administration of 5%, but not 0.5% (w/w) sodium saccharin in the diet significantly increased the incidence and extent of PN hyperplasia. This finding could be related to the induction of cancers in the rat urinary bladder by high levels of saccharin. Sodium ascorbate (5%). DL-tryptophan (5%) and allopurinol (0.02%) also significantly increased the extent of PN hyperplasia in the affected animals, but other test chemicals, such as acetazolamide (0.35%) and quercetin (5%) did not. The results with sodium saccharin and DL-tryptophan were consistent with previous findings and suggest that sodium ascorbate and allopurinol have promoting activities in urinary bladder carcinogenesis in rats. No correlation was found between the extent of crystalluria and promotion of preneoplastic lesions.     

Effects of different types of diet and sodium saccharin on proliferation at the limiting ridge of the rat forestomach

Sodium saccharin, at high doses in the diet, has been reported to cause hyperplasia of the forestomach (squamous portion of stomach), at the limiting ridge in F344 rats, in addition to its potential to induce proliferative effects on the urinary bladder epithelium. We have characterized this hyperplasia of the squamous epithelium of the forestomach at the limiting ridge in F344 and Sprague-Dawley rats given various doses of sodium saccharin for 4 to 95 wk. With increasing doses of sodium saccharin, the limiting ridge of the forestomach showed dose-related morphological changes: basal-cell hyperplasia, early papillary hyperplasia with basal-cell hyperplasia and papillary hyperplasia. Calcium saccharin in Prolab diet caused hyperplasia of the forestomach at the limiting ridge, similar to that caused by sodium saccharin. The severity of hyperplasia was influenced by the type of diet and by the strain of rats. AIN-76A diet without added sodium saccharin caused basal-cell hyperplasia in F344 rats, whereas Prolab, Purina and NIH-07 diets without added sodium saccharin had little or no effect on the forestomach. The effect of AIN-76A diet alone persisted through 95 wk of feeding without any evidence of tumour formation. In Sprague-Dawley rats, which appeared more sensitive to effects on the forestomach than F344 rats, Prolab 3200 and Purina diets without sodium saccharin caused basal-cell hyperplasia in more than half of the treated rats. The forestomach hyperplasia associated with AIN-76A or saccharin administration appears to be mild, limited in extent to the limiting ridge, and not associated with carcinogenesis.     

Dietary effects of ortho-phenylphenol and sodium ortho-phenylphenate on rat urothelium.

Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are pesticides used commercially in the food industry that have been shown to be carcinogenic to rat urothelium. Dietary administration of 1.25% OPP or 2.0% NaOPP caused increased incidences of urothelial hyperplasia and eventually caused tumors in male F344 rats, with NaOPP apparently having a more potent effect. In other studies, various sodium salts such as saccharin and ascorbate enhanced bladder carcinogenesis, although the acid forms of these salts did not. In studies with high dietary doses of these sodium salts, an amorphous precipitate was produced in the urine; precipitate formation was pH dependent. In previous experiments in which high doses of OPP were fed for up to 17 weeks, severe hyperplasia of the urothelium was produced, but without the formation of an urinary amorphous precipitate, calculi, or abnormal microcrystalluria. In addition, we found no evidence of OPP-DNA adduct formation in the urothelium. The present study was conducted to determine if feeding NaOPP * 4 H(2)0 to male F344 rats as 2.0% of the diet resulted in the formation of an amorphous precipitate in the urine, and if NaOPP caused an increased mineral concentration in the urine and/or kidneys. NaOPP administration produced a higher urinary pH than did OPP fed as 1.25% of the diet. Neither amorphous precipitate nor other solids were observed in the urine of the OPP or NaOPP-treated rats, and urinary calcium concentrations in the treated groups were similar to control. OPP and NaOPP had similar proliferative effects on rat urothelium after 10 weeks of treatment by light microscopy, scanning electron microscopy (SEM), and bromodeoxyuridine (BrdU) labeling indices. The results of this study indicate that formation of abnormal urinary solids is not part of the mechanism by which OPP or NaOPP exert their effects on the rat bladder epithelium.     

Effects of Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on the urinary bladder of male Wistar rats

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide widely used on agricultural crops such as soy, cotton and sugar cane. In a previous long-term study this herbicide exerted carcinogenic activity on the urinary bladder mucosa of male Wistar rats. In general, the genotoxic and mutagenic potentials of Diuron are considered to be negative. The present study aimed to evaluate the mode of action of Diuron on the urinary bladder mucosa of male Wistar rats. Six-week old male Wistar rats were fed pelleted Nuvilab diet mixed with Diuron at 125, 500 and 2500 ppm. As a positive control, 8.3% sodium saccharin (NaS) was fed in the diet. Preceding the sacrifice of the animals at the 20th week, urinary pH was measured and the genotoxic potential of Diuron was evaluated by the comet assay. Histological urothelial lesions in the urinary bladder and in the renal pelvis mucosa, cell proliferation/apoptosis evaluations, and scanning electron microscopy (SEM) of the urinary bladder mucosa were also performed. No DNA changes were found in urothelial or peripheral blood cells, and urinary pH was comparable to controls in all Diuron groups. In the urinary bladder urothelium, the incidence of simple hyperplasia (SH) by light microscopy was significantly increased (7/10; p<0.005) in the 2500 ppm Diuron group but not at the lower doses. By SEM, three of five animals treated with 2500 ppm Diuron showed urothelial cell necrosis and hyperplasia. In the renal pelvis, the incidence of SH was significantly increased in the Diuron 500 and 2500 ppm and in the NaS 8.3% groups. Cell proliferation was significantly increased in the Diuron 2500 ppm (p<0.05) and NaS 8.3% (p<0.05) groups. The results indicate that a high dietary concentration of Diuron is associated with urothelial necrosis and continuous regenerative cell proliferation that leads to urothelial hyperplasia.     

Alterations in the rat kidney associated with sodium saccharin feeding

Sodium saccharin has previously been demonstrated to induce hyperplasia and tumors of the urothelium of the rat urinary bladder. It was fed as 5% of the diet to male F344 rats for 2 years. In the present experiment, mild simple hyperplasia of the urinary bladder epithelium was again frequently observed, and a marked nodular and papillary hyperplasia of the urothelium of the kidney pelvis was also found in approximately half of the sodium saccharin-fed rats. This was infrequently associated with focal calcification of the renal papilla and pelvis. In contrast, rats fed sodium saccharin had a significantly reduced incidence of the interstitial nephritis frequently observed in older rats. No significant incidence was observed of lesions of tissues other than those of the urinary tract.     

Bladder freeze ulceration and sodium saccharin feeding in the rat: Examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.     

Chronic toxicity and carcinogenicity to the urinary bladder of sodium saccharin in the in utero-exposed rat

In utero-exposed Charles River CD strain rats, the offspring of parents treated from weaning through mating, gestation and lactation, were fed sodium saccharin chronically at dietary levels of 0, 0.01, 0.1, 1.0, 5.0, and 7.5%. Calcium cyclamate was fed as a reference compound at a level of 5.0%. Groups of 48 male and 48 female weanlings were placed on the same regimen as their parents. The study was continued until the number of survivors in a group fell to 20% with the last survivors being killed approximately 28 months after the first weanlings were selected for the chronic study. Serial sacrifices were performed at 14 and 18 months. Treatment-related effects were not observed in hematological values, organ weights, and survival. Average weaning weights were decreased in litters from parents receiving 5.0 and 7.5% sodium saccharin and 5.0% calcium cyclamate. Some of the initial weaning weight depression was overcome, but the rats on these dose levels had lower average body weights throughout the study. The incidence and types of neoplasms and nonneoplastic lesions, other than urinary bladder neoplasms and hyperplasia, were typical of the aged rat and were not treatment related. A significantly increased incidence of urinary bladder hyperplasia occurred in female rats that received 7.5% sodium saccharin, but the lesion was not morphologically precancerous. A significantly increased incidence of urinary bladder neoplasms occurred in the males fed 7.5% sodium saccharin. A total of 11 bladder neoplasms was observed in rats on study longer than 18 months; nine in the 7.5% saccharin group and one each in the control and 5.0% saccharin groups. There were nine neoplasms in males and two in females. Histologically, six of the neoplasms were classified as benign and five as malignant. All the malignant neoplasms occurred in the 7.5% saccharin group. The occurrence of the urinary bladder neoplasms could not be related to such predisposing factors as gross calculi, parasites, or hyperplasia.     

Comparison of the bladder response to indole and sodium saccharin ingestion by male rats

To ascertain whether the bladder mass increase and epithelial hyperplasia induced by 5% dietary sodium saccharin (NaS) in short-term experiments with rats are caused by increased urinary excretion of indican associated with this treatment, the responses of the urine and bladder induced by 1.5% indole (Id) ingestion were compared with those induced by 5% NaS and 1.5% Id + 5% NaS. Id and NaS, when fed alone, produced equivalent increases in bladder mass and both compounds induced epithelial hyperplasia, but Id ingestion was associated with much greater urinary indican excretion (5 mg/g diet ingested) than was NaS (0.3 mg/g diet ingested). When Id and NaS were ingested together, the bladder mass increase was additive, but the epithelial hyperplasia was not exacerbated over that observed with each alone, and the urinary indican was equivalent to that produced by Id alone. These findings suggest that a high level of urinary indican excretion is associated with an increase in bladder mass and epithelial hyperplasia (Id treatment) but indicate that the relatively low urinary indican level obtained by NaS feeding alone is unlikely to be responsible for the bladder responses noted with this compound.     

The Effect of Lifetime Sodium Saccharin Dosing on Mice Initiated with the Carcinogen 2-Acetylaminofluorene

The Effect of Lifetime Sodium Saccharin Dosing on Mice Initiatedwith the Carcinogen 2-Acetylaminofluorene. FREDERICK, C. B.,DOOLEY, K. L., KODELL, R. L., SHELDON, W. G , AND KADLUBAR,F. F. (1989). Fundam. Appl. Toxicol 12,346–357. sodiumsaccharin has been reported to promote the development of urinarybladder tumors in rats following low doses of several carcinogens.To evaluate the generality of this effect between species, aninitiation-promotion study was conducted in mice. Weanling femaleBALB/c mice were initiated with 200 ppm dietary 2-acetylaminofluorenefor 90 days. Following a 2-week period of control diet, saccharinwas administered at 0, 0.1, 0.5, 1.0, and 5.0% in the diet forthe remainder of the 132-week study. An elevated incidence ofpersistent bladder transitional cell hyperplasia and a low incidenceof urothelial and hepatocellular tumors indicated that theseorgans achieved an adequate dose of the initiator. However,sodium saccharin dosing did not result in an increased incidenceof tumors in either the bladder or liver and is therefore notconsidered to be a promoter of carcinogenesis at these sitesin the mouse. Furthermore, sodium saccharin exhibited a modestinhibitory effect on the rate of development of lymphomas inboth initiated and noninitiated animals. Interspecies differencesin the bladder tumorigenic effect ofsodium saccharin and theirassociation with differences in urinary tract physiology arediscussed.     

Histopathological evaluation of rat urinary bladders from the IRDC two-generation bioassay of sodium saccharin

Bladder tissues from rats treated with sodium saccharin in a two-generation dose-response study, in which 1.0% in the diet had been identified as the no-effect level for primary bladder cancer, were re-examined without knowledge of the test groups or the previous histopathological findings. Compound-related increases in the incidences of hyperplasia were found at the 6.25 and 7.5% saccharin levels (the highest dietary levels tested). There were also compound-related increased in the incidence of transitional-cell papillomas and/or carcinomas at the 4.0, 5.0, 6.25 and 7.5% levels. There was a slight increase in the incidence of transitional-cell carcinomas at the 3.0% level, but this was not statistically significant (P = 0.25). No compound-related effects were evident at the 1.0% level. All tumours were well to moderately differentiated and there was no increase in mean grades of severity or anaplasia for any proliferative lesion.     

Induction of reversible urothelial cell hyperplasia in rats by clorsulon, a flukicide with weak carbonic anhydrase inhibitory activity.

Clorsulon, a flukicide registered for use in treating Fasciola hepatica infections in cattle, has induced urinary bladder urothelial cell hyperplasia in rats at oral doses of 30 mg/kg/day or more. Despite previous testing at doses above this threshold, this lesion had not been found in subchronic or chronic toxicity studies in rats. After ruling out the presence of a contaminant as the causative factor in producing this lesion, a study was conducted in which clorsulon increased the pH and altered the electrolyte composition of urine, consistent with its weak carbonic anhydrase inhibitory activity. The acid/base balance of the diet markedly affected the threshold for induction of the urothelial cell hyperplasia: acidification by addition of 5% ammonium chloride to the diet reduced the incidence and severity. In additional studies it was found that the urothelial cell hyperplasia was most pronounced after 1 week of treatment compared with daily exposure for either 5 or 15 wk. The reversibility of the hyperplasia despite continued treatment confirms that the hyperplasia is not a preneoplastic lesion, a conclusion supported by negative studies of carcinogenicity in rodent bioassays of clorsulon and other drugs with carbonic anhydrase inhibitory activity.     

Some changes in gastro-intestinal metabolism and in the urine and bladders of rats in response to sodium saccharin ingestion

In rats fed sodium saccharin in the diet changes in urine composition, increased bladder-tissue mass and, in males only, an accumulation of minerals in the bladder tissue have been observed. In this report evidence is presented that indicates that these changes are a consequence of the effects of sodium saccharin in the gastro-intestinal tract and are not due to systemic sodium saccharin. Sodium saccharin has been shown to inhibit gastro-intestinal enzymes that digest carbohydrates and proteins and to increase caecal absorption of mineral ions. The significance of these findings to saccharin-associated bladder tumorigenesis is discussed.     

The effect of various saccharin forms on gastro-intestinal tract, urine and bladder of male rats

Sodium saccharin, potassium saccharin, calcium saccharin and the free acid when fed to young male rats at a level of about 200 mumol/g diet all produced an equivalent increase in the caecal enlargement indicating that this phenomenon was due to the saccharin ion and not the accompanying cation. The sodium and potassium salts caused greater polydipsia and polyuria than the calcium or free acid forms. Simple hyperplasia of the bladder was noted in the rats ingesting the sodium and potassium salts but not in those ingesting the calcium or free acid forms. The difference in urine and bladder response to the salt forms is not attributable to the difference in the total urinary saccharin or the urinary concentration of saccharin. These results suggest that excess water absorption from the lower bowel and the concomitant bladder responses are dependent upon monovalent cation absorption but independent of saccharin absorption.     

Effect of inherent urine output on the response of male rats to 7.5% dietary sodium saccharin

Young male rats were preselected as high urine (57 g/kg body weight) or low urine (35 g/kg) voiders and were fed a diet containing 7.5% sodium saccharin (NaS) for 10 wk. Urine output was found to be a stable characteristic and high urine output was associated with increased water and feed consumption and increased weight gain. Rats responded in a very similar fashion to 7.5% dietary NaS regardless of their inherent urine output. NaS ingestion was associated with increases in water consumption, caecal mass and urine volume. Among rats that had ingested 7.5% dietary NaS for 10 wk there was a high incidence (12/20) of bladder epithelial hyperplasia. The results are discussed with regard to the concept that increased urine output is an important factor in NaS-induced bladder tumours.     

Lack of bladder carcinogenicity of dietary sodium saccharin in analbuminaemic rats, which are highly susceptible to N-nitroso-n-butyl-(4-hydroxybutyl)amine

A previous 1-wk study measuring the agglutinability of bladder epithelial cells by concanavalin A had suggested a high susceptibility of analbuminaemic rats to bladder carcinogenesis by sodium saccharin (Honma et al., Cancer Letters 1983, 19, 7). A long-term study was conducted to confirm the results of this short-term assay. Sodium saccharin was administered at 5% in the diet to groups of 35 male analbuminaemic rats and 36 male rats of the Sprague-Dawley strain. No bladder carcinomas nor precancerous lesions were observed in any of the rats exposed to sodium saccharin. The discrepancy between the results of the agglutination assay and the long-term experiment might be attributable to a sharp increase in urine volume in analbuminaemic rats during the first week of treatment. The resultant distension of the bladder wall might make the epithelial cells more susceptible to concanavalin A agglutination. The results indicated no demonstrable bladder carcinogenicity of sodium saccharin in analbuminaemic rats and excluded the possibility that these particular mutant rats could be useful animals for shorter-duration screening for bladder carcinogens.     

Long-term toxicity of ortho-toluenesulfonamide and sodium saccharin in the rat

In a two-generation lifetime feeding study, 50 male and 50 female weanling Sprague-Dawley rats were included in each of the following dietary treatment groups: control; 2.5 mg ortho-toluenesulfonamide (o-TS, more than 99.9% pure)/kg/day; 25 mg o-TS/kg/day; 250 mg o-TS/kg/day; 250 mg o-TS/kg/day with 1% NH₄Cl in the drinking water: or 5% sodium saccharin, which contained no detectable amount of o-TS. After 3 months on test, the F₀ rats were bred and 50 pups of each sex, from every group, were weaned onto the parental diets which both generations received for their lifetime. Rats from both generations fed diets providing 250 mg o-TS/kg with 1.0% NH₄Cl in the drinking water or containing 5% sodium saccharin had decreased growth rates, but only the former two had lowered feed consumption. There were no treatment-related effects upon reproduction, longevity, or hematological parameters. The animals were free of the bladder parasite Trichosomoides crassicauda. A few animals developed grossly visible bladder and kidney stones but the incidence of these was not treatment related. The incidence of bladder tumours in male rats fed the 5% saccharin diet was significantly increased in both generations compared to their respective control groups. An evaluation of individual feed consumption data for animals fed saccharin-containing diets did not reveal any statistical difference between the amount of feed consumed by animals which had bladder tumors and those that did not. The incidence of bladder tumors in the o-TS-treated groups and in the female rats fed the 5% saccharin diet was not significantly different from that in control animals.     

Multi-stage carcinogenesis in the urinary bladder

Sodium saccharin has been shown to be a promoting substance for urinary bladder carcinogenesis in the rat following initiation with N-methyl-N-nitrosourea, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), or N-butyl-N-(4-hydroxybutyl)nitrosamine. It has been shown to have many of the properties of promoting substances in other animal models, such as the mouse skin; it lacks mutagenic activity, induces hyperplasia in the target tissue, and does not bind DNA. It has recently been demonstrated to be co-carcinogenic for the rat bladder. It has also been shown that the administration of sodium saccharin during the regenerative hyperplasia observed after freeze ulceration or cyclophosphamide administration resulted in the induction of bladder tumours, even without pre-initiation with FANFT or other known initiating substances. This model appears to be analogous to the administration of sodium saccharin to animals with a rapidly proliferating bladder mucosa as occurs in utero during the two-generation carcinogenesis experiments and in the pellet-insertion experiments in which a cholesterol pellet containing sodium saccharin is inserted into the bladder. To enhance our understanding of the complex interaction of the many variables involved in two-stage bladder carcinogenesis, a stochastic model has been formulated based on long-term carcinogenicity and in vivo tissue kinetic studies. This model indicates the importance of cell proliferation and the development of hyperplasia in carcinogenesis.     

A review and biological risk assessment of sodium saccharin.

Dietary sodium saccharin is associated with bladder tumors when fed at high levels to the male rat. Under these conditions urinary pH, sodium concentration, and volume are elevated and proliferative changes are present in the urothelium. Extensive epidemiological studies have shown that saccharin does not increase the risk of bladder cancer in humans and laboratory investigations have shown that sodium saccharin is not mutagenic and does not bind to DNA. Recent research indicates that the urothelium in male rats is damaged under conditions of high urinary pH and sodium levels by a mechanism that involves alpha 2u-globulin and possibly silicate crystalluria. These studies and their implications for human health risk are reviewed.