Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats

Samuel M. Cohen1,2,5, Lora L. Arnold1, Martin Cano1, Masahiro Ito1, Emily M. Garland1,4 and R.Anthony Shaw3

1 Department of Pathology and Microbiology,
2 The Eppley Institute for Research on Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198–3135 and
3 National Research Council of Canada, Institute for Biodiagnostics, Winnipeg, Manitoba R3B 1Y6, Canada


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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.

Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; BrdU, bromodeoxyuridine; FANFT, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide; HPLC, high performance liquid chromatography; MNU, N-methyl-N-nitrosourea; OTS, O-toluene sulfonamide.


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In 1970, sodium saccharin, incorporated into cholesterol pellets and surgically implanted into mouse bladder, was shown to increase the incidence of bladder tumors (1). Although subsequent studies demonstrated that this tumorigenic effect was due to the pellet itself rather than the sodium saccharin (24), this study raised concern about the safety of saccharin as an artificial sweetener and food additive (5) and led to additional testing of its carcinogenicity. This resulted ultimately in the findings presented as an abstract in 1974 (6) indicating that sodium saccharin fed at high doses was a bladder carcinogen in rats when administered over two generations. This study involved administration of the compound to the male and female F0 parental generation prior to mating, then to the dams during gestation and lactation, and then to the weaned pups for the remainder of their lives for up to 2–2.5 years. This initial report was confirmed by three subsequent studies (79) all indicating that sodium saccharin fed at high doses in the two-generation protocol increased bladder cancer incidence in rats. The tumorigenic response was greater in male than in female rats. Administration of the same doses of this compound to rats beginning at 35 days of age or later (traditional 2-year bioassays), however, did not significantly increase the incidence of bladder tumors (8,1015).

In contrast to the lack of carcinogenicity of this compound when administered in a standard 2-year bioassay beginning at 5 weeks of age or greater, sodium saccharin produced a significant increase in the incidence of tumors when administered to adult rats after brief exposure to known bladder carcinogens such as MNU (10,11,16,17), FANFT (1214,18,19), BBN (20,21) or cyclophosphamide (14), and after freeze ulceration (14,15) of the bladder. In addition, sodium saccharin administered to adult rats produced mild superficial urothelial cell cytotoxicity with consequent mild regenerative hyperplasia (2225). The increase in cell proliferation has been demonstrated by light microscopy, scanning electron microscopy and by labeling index studies utilizing either tritiated thymidine (22,23) or BrdU pulse labels (24,25).

Saccharin is generally considered to be non-genotoxic (26). In most screens for genotoxicity it is negative. However, positive results have occasionally been observed in chromosomal rearrangement and related assays, but only at high levels of exposure (26,27). Other sodium and potassium salts, such as NaCl or KCl, are also positive in these assays (27), and it has been suggested that the genetic effect is secondary to the osmotic changes produced by high concentrations of these salts rather than being due directly to the chemical itself (28). In addition, saccharin is a moderately strong organic acid [pKa ~2.0 (29)], it is not metabolized (30), and at intracellular pH it is present nearly entirely as an anion, which would not be expected to react with DNA. Lutz and Schlatter (31) found no evidence of DNA–saccharin binding when administered at high doses to rats. Zukowski et al. (32) also demonstrated that high doses of sodium saccharin fed to rats did not produce unscheduled DNA synthesis in the bladder urothelium in an in vivo–in vitro assay.

Hasegawa and Cohen (33) found that the salt form of saccharin administered significantly influenced whether the rats developed a proliferative response to the saccharin, although the doses were essentially the same with the different forms. The proliferative response to sodium saccharin was greater than with potassium saccharin, and the response with calcium saccharin was marginally, but not statistically significantly, increased. Acid saccharin was without effect on the bladder epithelium. Although the urinary concentrations of saccharin were the same following feeding with any of these forms of saccharin, the other constituents of the urine varied considerably between the different groups as anticipated given the physiological response to the large load of either sodium, potassium, calcium or hydrogen ions. Thus, the proliferative response following high doses of saccharin in the diet is not dependent solely on the urinary concentration of the saccharinate ion.

A possible explanation for the differences in urothelial response to the oral administration of different saccharin salts is that the various ionic changes in the urine alter the structure of the saccharinate ion. However, this was examined by Williamson et al. (29) using various NMR analyses and was found not to be the case. With variations in the ionic milieu comparable to the wide variations in urinary concentrations following administration of these different saccharin salts, there was no difference in the structure of the saccharinate ion. These studies and others suggested that the proliferative response and tumorigenicity of saccharin were probably due to an indirect process rather than interaction of saccharin itself with the bladder epithelium. No evidence of saccharin binding to a cell receptor has been observed (34).

In the 1970s, the Canadian government undertook a detailed study of the carcinogenicity of sodium saccharin in a two-generation protocol in rats, and also evaluated the possibility that its carcinogenic activity was due to a contaminant, OTS (8). It was conclusively demonstrated that OTS was not responsible for the bladder carcinogenicity of saccharin in the rat. Subsequent studies on the proliferative and carcinogenic effects of saccharin used sodium saccharin synthesized by the Maumee procedure, which does not result in OTS as a contaminant, rather than the Remsen–Fahlberg method originally used, which does yield OTS as a contaminant.

During the course of the Canadian bioassay study by Arnold et al. (8), it was noted that rats fed high doses of sodium saccharin in the diet developed cloudy urine. The investigators were able to show that the cloudiness was due to the formation of a urinary precipitate, and they demonstrated that the precipitate contained relatively small amounts of protein and small amounts of saccharin (35). Further analyses were not reported. The presence of this precipitate in the urine was confirmed by West and Jackson (36), but no analysis of its composition or its relationship to the carcinogenicity of saccharin undertaken. In the mid 1980s we rediscovered the presence of this precipitate in the urine of animals fed high doses of sodium saccharin, and have since pursued various analytical and correlative studies of its relationship to the proliferative and carcinogenic activities of sodium saccharin in the rat (37,38).

Other sodium salts behave similarly to sodium saccharin when fed at high doses in the diet to rats. Including similar induction of superficial urothelial cell cytotoxicity, regenerative hyperplasia and tumorigenicity when administered following a short exposure to a genotoxic bladder carcinogen, BBN being the most frequently used carcinogen (18,3943). A precipitate, morphologically similar to that following sodium saccharin feeding, forms in the urine of animals fed these other sodium salts (38).

Initial analyses of this precipitate in our laboratory suggested that silicon-containing substances (likely to be silicates) were a significant component and might be related to the biological effects of the precipitate (37). It is known that a variety of silicates concentrated in the urine can produce crystalline material that is cytotoxic to the bladder epithelium leading to consequent regeneration, and ultimately, to the development of bladder tumors in various experimental and domestic animals (4446). Although subsequent studies in our laboratory have confirmed the presence of silicon, they suggest that it is only a minor component of the precipitate. The contribution of silicon-containing substances to the cytotoxic effects following feeding of sodium saccharin or other sodium salts remains unknown.

Since the presence of this precipitate appears to be critical for the proliferative and tumorigenic effects of these sodium salts, we have continued our investigations on its composition and on the factors that influence its formation in the rat. The results of these investigations are reported here, together with a discussion of the supporting data from other studies indicating the role this precipitate has in producing the urothelial effects in the rat bladder following feeding of sodium saccharin or other sodium salts at high doses.


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Animal treatments
F344 rats were obtained from Charles River Breeding Laboratories (Kingston, NY or Raleigh, NC) at 4 weeks of age, weighing ~40–50 g. They were maintained and quarantined for ~1 week and then started on treatment with the respective diets for 4–26 weeks, depending on the specific experiment. Groups of treated and control animals were maintained continuously in the laboratory for the acquisition of fresh voided urine for precipitate and urinary chemical analyses. The treatments and protocols of these experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. The rats were maintained in a level 4 barrier facility with five rats per cage in polycarbonate cages (Lab Products, Maywood, NJ) on corncob bedding (Anderson, Maumee, OH). Room temperature was maintained at 71 ± 5°C and a relative humidity of 30–70% on a 12 h light/12 h dark cycle with the changes occurring at 0600 and 1800 h. The rats were weighed at the beginning of each experiment and then every 2–6 weeks thereafter. Food and water consumption was measured at the same intervals. Prolab 3200 diet (Agway, St Mary's, OH) was used for all experiments except where stated, and the diets were mixed and pelleted by Dyets (Bethlehem, PA).

Sodium saccharin was received as a gift from PMC Specialties Group (Cincinnati, OH). Sodium ascorbate and ammonium chloride were purchased from Sigma (St Louis, MO). The purity of saccharin was verified by NMR analysis. Sodium ascorbate and ammonium chloride were used as received, relying on the company's chemical analyses as reported with the shipment. Dietary levels of the chemicals were determined by methods previously described (47,48).

Processing of tissues
At the end of each experiment the animals were killed with an overdose of Nembutal (50 mg/kg i.p.). The bladder and stomach were inflated in situ with Bouin's fixative, ligated, immediately removed, and placed in the same fixative, and then washed with 70% ethanol. The kidneys were also removed and placed in 10% phosphate buffered formalin (pH 7.4). All tissues were processed for light microscopy. The bladders were also processed for scanning electron microscopic evaluation as described previously (23). For some studies, 1 h prior to terminal sacrifice the rats received an i.p. injection of BrdU (100 mg/kg) for determination of the labeling index of the epithelial cells. A slice of stomach, through the limiting ridge, was included in the cassettes with the slices of bladder tissue to be processed for light microscopy; the stomach served as a positive marker of labeling with the BrdU.

Urine collection
Because of variability occurring secondary to diurnal variations and changes that can occur secondary to long term collections (49), fresh voided urine specimens were used for most of the analyses. The procedure for obtaining fresh voided urine could not be performed too frequently as this procedure itself can produce changes in the urine and in the urothelium of the rat (50). Fresh voided urine specimens were collected by holding the rats directly over a 1.5 ml conical plastic test tube and firmly squeezing the back of the neck. This resulted in urine samples from rats ~90% of the time, with a sample size of 10–1000 µl. Urine specimens were collected between 0700 and 0900 h. Depending on the experiment, either whole urine was used for chemical analyses or the specimen was centrifuged at 7000 g and the supernatant aspirated for chemical analyses.

Processing of precipitate for analysis
To examine the precipitate morphologically, it was transferred to 22-µm filters (Millipore, Bedford, MA) which were glued to aluminum specimen stubs and then processed for scanning electron microscopic observation as described previously (38). The specimens were examined in a 515 Phillips Scanning Electron Microscope (Phillips, Eindhoven, The Netherlands) with an attached Kevex Micro-X 7000 X-ray energy dispersive spectrometer (Kevex, Haywood, CA) at 20 kV voltage. The specimens were examined uncoated to provide a true spectrum without a heavy metal peak overlap.

For chemical analyses of the precipitate, samples from individual rats were used whenever possible although on occasion samples were pooled between various animals for specific analyses as indicated below. For the chemical analyses, the precipitate was wicked dry after centrifugation, and then resuspended in 100 µl 100 mM HCl. Silicon composition was analyzed by atomic absorption utilizing a Perkin Elmer Model 1100 Atomic Absorption spectrometer (Norwalk, CT) with a wavelength of 251.6 nm. Calcium (51), phosphorus (52) and magnesium (53) were analyzed using micro methods (Sigma). Protein was analyzed utilizing SDS–PAGE for total protein and western blot analyses for {alpha}2u-globulin and albumin. The antibody for {alpha}2u-globulin was purchased from Hazelton (Vienna, VA); the antibody for albumin was purchased from The Binding Site (San Diego, CA). Sulfated mucopolysaccharides were analyzed using a colorimetric method (54) based on binding of 1,9-dimethylmethylene blue (Biocolour, Belfast, Ireland). Analysis for specific mucopolysaccharides was performed using an HPLC method (55). The HPLC system consisted of a Waters (Milford, MA) 510 pump and WISP 712 injector and a Perkin Elmer LS-5B Luminescence Spectrometer. The pump was operated at a flow rate of 0.8 ml/min. Separations were carried out on a Nucleosil SB 5 anion exchange column (4.6x150 mm, 5 µm particles) for sulfated glycosaminoglycans and on a Hypersil APS weak anion exchange column (4.6x250 mm, 5 µm particles) connected to the Hypersil ODS RP-C 18 column for unsulfated glycosaminoglycans. Saccharin (47) and ascorbate (48) were analyzed as previously described.

Urinary chemistries
Urinary silicon (56) concentration, calcium (57), phosphorus (52), magnesium (58), sodium (59), potassium (60), chloride (61) and creatinine (62) were performed on whole urine or supernatant (no differences were observed between these) utilizing methods previously described. All but silica were assayed on an Ektachem 700 (Kodak, Rochester, NY). These could all be done on a relatively small sample volume (10 µl), however, occasionally not all could be done on the same sample. Osmolality required a larger sample (100 µl) and was determined by freezing point depression using a 3MO Advanced Micro-Osmometer (Norwood, MA).

Infrared analysis
Infrared spectra were collected using a Digilab FTS-40A spectrometer (Bio-Rad Laboratories, Cambridge, MA), equipped with a Split-Pea sampling accessory (Harrick Scientific, Ossining, NY). This accessory measured the attenuated total reflection spectrum of the sample, pressed into close contact with the surface of the silicon optical element. The infrared beam probed a circular area of ~300 µm diameter, penetrated to a depth of ~4 µm. All spectra were acquired at a resolution of 2 cm–1, co-adding 256 interferograms.

To guide the interpretation of the precipitate infrared spectra, infrared spectra were also measured for several reference compounds that were considered as possible constituents. These included urea, silica, sodium saccharin, sodium ascorbate, mono-, di- and tribasic calcium phosphates, sodium chondroitin sulfates B and C, sodium hyaluronate, sodium heparin sulfate, {alpha}2u-globulin, albumin, uric acid, ascorbic acid, creatinine, oxalic acid and citric acid.

Immunoperoxidase staining
Paraffin-embedded sections were immunohistochemically stained utilizing a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) for BrdU (63). Antibody to BrdU was obtained from Boehringer Mannheim (Indianapolis, IN) and was utilized at a dilution of 1 to 50. Anti-{alpha}2u-globulin (Hazelton) at a dilution of 1 to 100 000 was used to stain for {alpha}2u-globulin according to the procedure of Dietrich and Swenberg (64).

Collection of urine from renal pelvis
Urine was collected from the upper portion of the ureter of rats for determination of the presence or absence of the amorphous precipitate in the upper urinary tract, in addition to its presence in urine in the bladder. Rats were anesthetized with Metofane. Normal saline was infused either through the jugular vein or femoral artery prior to collection of specimens. The ureters were dissected, severed and allowed to drain directly onto 22-µm filters.

In vitro effects of calcium phosphate
To evaluate the cytotoxic nature of calcium phosphate, C8 rat bladder epithelial cells (65) were grown to 70–80% confluency in 25 cm2 flasks in Medium 199 (Gibco, Grand Island, NY) and Ham's F-12 (1:1) (Gibco) with 5% fetal bovine serum. C8 cells are immortalized bladder epithelial cells, but are not tumorigenic when transplanted back to syngeneic rats. The cells do not show terminal differentiation. This medium contains 0.7 mM calcium and 1.1 mM phosphorus. The medium was then supplemented with 5 mM calcium phosphate which led to the formation of precipitate in the medium. Cell viability was examined microscopically and by trypan blue exclusion at 1, 6 and 24 h after exposure began.


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Precipitate analysis
The precipitate is a white, flocculent material that sediments upon centrifugation. By scanning electron microscopy it has a typical appearance (Figure 1Go) regardless of the circumstances of animal treatment leading to its formation in the urine. The flocculent material is in addition to the usual magnesium ammonium phosphate crystal (Figure 2Go) that is present in control rat urine. The precipitate is rarely present in the urine of control rats. It can be distinguished from deteriorated magnesium ammonium phosphate crystals and from cellular material partly by its more granular appearance and definitively by its X-ray energy dispersive spectrum.



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Fig. 1. Scanning electron microscopic appearance of amorphous urinary calcium phosphate-containing precipitate from a rat fed 7.5% sodium saccharin; x263.

 


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Fig. 2. Scanning electron microscopic appearance of urinary magnesium ammonium phosphate crystals from a control rat; x600.

 
By using energy dispersive X-ray spectroscopy, several elements are detected (Figure 3Go); the most prominent are calcium and phosphorus. The phosphorus peak appears to be related to its presence as a phosphate (see below). Other elements that are present are magnesium, silicon and sulfur. Lower atomic weight elements, such as carbon, nitrogen, oxygen and sodium, are not detectable by this method. The extent of the presence of the elements detected by energy dispersive X-ray spectroscopy other than calcium and phosphorus is relatively minor.



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Fig. 3. X-ray energy dispersive spectroscopy of urinary calcium phosphate-containing precipitate.

 
The infrared spectra of urinary precipitates from rats fed sodium saccharin could be reconstructed on the basis of five major constituents (Figure 4Go). The clearest signatures correspond to saccharin and urea; each of these compounds exhibits a unique infrared `fingerprint' that is clearly discernible in the spectrum of the precipitate. More subtle precipitate absorptions were best accounted for by postulating the presence of tribasic phosphate, silicate and sodium chondroitin sulfate. Both tribasic (but not mono- or dibasic) phosphate and silicate show very simple absorption spectra that account for the diffuse precipitate absorption in the 950–1200 cm–1. Finally, these assignments are consistent with (i) the X-ray spectroscopic evidence suggesting high calcium and phosphorus levels, as well as the presence of silicon, and (ii) other evidence (Alcian blue staining and chemical analyses by colorimetric methods) indicating substantial amounts of acid mucopolysaccharide (see below, HPLC analysis).



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Fig. 4. Infrared spectra of various precipitate specimens and pure chemicals.

 
The infrared spectrum of urinary precipitate from animals fed sodium ascorbate revealed no ascorbate and lower total mucopolysaccharide levels than found in precipitate from rats fed sodium saccharin (Figure 4Go). In addition, the peak at 995 cm–1, assigned to tribasic phosphate, is much more pronounced in the precipitate spectra for ascorbate-fed rats than in the spectra from saccharin-fed rats.

Infrared spectroscopy is unsuitable for the detection of trace components in the precipitates. Although the five components discussed here are sufficient to reproduce all major features of the precipitate spectra, we cannot rule out the presence of other species in concentrations <5%.

Specific quantitative analyses, for various chemicals in the precipitate, show that the major inorganic components were calcium and phosphate. However, the amounts varied considerably. The inorganic components of the precipitate usually accounted for 10–40% of the material, with ~85% of the inorganic material being calcium phosphate. Magnesium was also present to a variable extent. This correlated well with the presence of magnesium ammonium phosphate crystals in the urine. Extraction of the precipitate with freon 113 (SPI Supplies, West Chester, PA) removed the crystals from the sediment and also tended to eliminate the magnesium peak by X-ray energy dispersive spectroscopy and by chemical analysis. However, we cannot completely exclude the possibility that magnesium is present in the precipitate in addition to the crystals, although clearly calcium and phosphate are the major inorganic components. No other differences were observed between freon-extracted precipitate compared with the precipitate not extracted. In contrast, the sediment from untreated control rats contained primarily magnesium and phosphate, representing the magnesium ammonium phosphate crystals. Calcium was usually present as <5% of the control sediment, and often <1%.

Silicon-containing material was also consistently detected in the precipitate as analyzed by atomic absorption. However, it tended to be a relatively minor component of the precipitate, and it was at similar levels in the sediment obtained from control rats and from saccharin-treated rats.

Extracts of the precipitate also demonstrated small amounts of saccharin (from animals fed saccharin), although this represented, consistently, a very small percentage of the total precipitate. Levels of ascorbate detected in the precipitate of animals fed sodium ascorbate were similar to the levels found in urinary sediment from control rats.

Although protein did not appear to contribute prominently to the infrared spectrum, small amounts of protein could be detected by extraction of the precipitate and chemical analysis. Also, we were able to specifically identify, by western blot analyses, that the two major proteins present in the precipitate were {alpha}2u-globulin and albumin.

A large amount of organic material was consistently present in the precipitate. The amount of sulfur present as saccharin is unlikely to explain the amount of sulfur that was detected by X-ray energy dispersive spectroscopy. Also, similar levels of sulfur were detected by X-ray energy dispersive spectroscopy in the precipitate from rats fed high levels of sodium ascorbate. We therefore evaluated the possibility of the sulfur being due to mucopolysaccharides in the precipitate. Initially, staining of the precipitate with Alcian blue (66) at pH 2.5 demonstrated that there was a large amount of acid mucopolysaccharide present. HPLC analysis of the precipitate from rats fed sodium saccharin showed chondroitin sulfate to be the major mucopolysaccharide present. No other mucopolysaccharides were detected in the precipitate because they were not present, or were present in amounts below the detection limit of the methodology (2.5 µg/ml).

Urine analyses
In rats administered either sodium ascorbate or sodium saccharin, the changes in urine were similar to those previously described, including the decrease in osmolality because of the increased urinary output. This was associated with decreases in urinary potassium, chloride, total protein, urea, and creatinine. However, when the sodium salt was administered, urinary sodium tended to increase. Against the dilutional effect, the concentrations of calcium, phosphate, and usually magnesium, tended to be the same or increased compared to control urine. When normalized to creatinine concentration, there were marked increases in the excretion of calcium and phosphate, and usually magnesium. Silicon concentrations tended to be similar in the urine of both treated and control rats.

The amount of total mucopolysaccharides in the urine for control and sodium saccharin-treated rats was 0.2 ± 0.0 and 0.4 ± 0.0 mg/ml, respectively. The major mucopolysaccharide was chondroitin sulfate (0.1 and 0.3 mg/ml in urine from control and treated rats, respectively). Small amounts of heparin sulfate were also detectable.

Total urinary protein tended to be decreased because of the overall dilution of the urine, but remained at extremely high levels as is typical of rat urine. The males had significantly more protein than females, and the difference was largely due to {alpha}2u-globulin. The amounts of {alpha}2u-globulin and albumin were similar proportionally in treated versus control rat urine.

In rats administered sodium saccharin, similar to other forms of saccharin (33), the urinary concentration of saccharin was directly proportional to the amount administered in the diet, reaching levels as high as 300 mM in the urine of rats fed 7.5% sodium saccharin in the diet. In contrast, the urinary concentration of ascorbate (ascorbate plus dehydroascorbate) was significantly lower (2.7 mM) than saccharin following comparable doses of sodium ascorbate in the diet. Nevertheless, there was a clear dose response for urinary ascorbate following administration of sodium ascorbate at levels 1.0–7.0% of the diet.

Male versus female
The concentration of saccharin and the other urinary parameters that were measured were similar in male compared with female rats except for total protein (Table IGo). In contrast, the frequency and the amount of precipitate appearing in the urine was significantly less in female rats than in male rats, although it was present in female rat urine administered high doses of sodium saccharin.


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Table I. Twenty-four hour urine chemistries during week 6 of treatment with 7.5% sodium saccharin
 
Dose response
Although we have not been able to quantify the amount of precipitate in the urine, an estimate was made measuring the optical density of the urine at 620 nm. This method is not sensitive but gives a general evaluation of the cloudiness of the urine that indicates the presence of solids in the urine. Utilizing that method, a no-effect level occurred at 1% sodium saccharin in the diet. In addition, at the dose of 1% sodium saccharin in the diet, there was no precipitate present in the urine following examination by scanning electron microscopy, a very sensitive method for detecting precipitate if present at all. At the dose of 3% sodium saccharin in the diet, there was occasionally precipitate present, but in relatively small amounts. The precipitate was consistently present at high doses, with a significant increase in optical density at a dose of 7.5% of the diet. Similar observations have been made with sodium ascorbate, although a small amount of precipitate was detected in five of seven rats fed sodium ascorbate even at a dose of 0.91% of the diet and heavy amounts in six of seven rats fed sodium ascorbate as 2.73% of the diet (67).

In vitro cytotoxicity
Calcium phosphate is an essential ingredient of any tissue culture medium, including urothelial cells. The medium that we have routinely used in our laboratory [Medium 199 and Ham's F-12 (1:1)] has a calcium concentration of 0.7 mM. When 5 mM calcium phosphate is added (total concentration 5.7 mM) calcium phosphate precipitates from the medium and settles onto the urothelial cells. After 1 h there are signs of cytotoxicity, including decreased cell number. Toxicity is greatly increased by 6 h, and most of the cells died by 24 h (Table II). At a concentration of 25 mM, the results are even more profound. There was no visual change in pH based on color of the medium. No other constituents of the media were varied, however, levels were not measured.

Other observations
Calcium phosphate-containing precipitate was observed in the urine of male rats administered 7.5% sodium saccharin in the diet beginning sporadically as early as 1 day after the administration of the chemical. It was more frequently present by 3 days, and was usually present in the urine by 7 days and subsequently. The presence and the amount of precipitate varied between rats at a given point in time and in the same rat at different times. This continued as long as the chemical was administered in the diet up to 26 weeks of administration.

The urothelial changes following administration of high doses of sodium saccharin in the diet are seen in the renal pelvis, ureters and urinary bladder (68,69). We have repeatedly been able to demonstrate the presence of the calcium phosphate-containing precipitate in fresh voided urine of rats administered sodium saccharin, and we have demonstrated its presence in the urine taken directly from the urinary bladder of rats. Studies to determine if a calcium phosphate-containing precipitate was present in the upper urinary tract of sodium saccharin-treated rats were inconclusive. We obtained urine from the upper ureters, presumably representing urine from the upper urinary tract including the renal pelvis. In the urine we found evidence of the same calcium phosphate-containing precipitate seen in the urine voided by treated rats and in the urine obtained from the urinary bladder in two samples from a sodium saccharin-treated rat. However, it was also present in one ureteral sample obtained from a control rat.

In rats fed 7.5% sodium saccharin in the diet, staining of the bladders and kidneys for {alpha}2u-globulin using an antibody to the protein showed no evidence of the protein in the urothelium of the renal pelvis, ureter or urinary bladder. As a positive control on the same tissue sections, it was present in the cells of the proximal tubules, similar to that demonstrated by numerous investigators previously (64).


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Administration of high doses of sodium salts produces an increase in urothelial proliferation in rats (2225). The proliferation represents a regenerative response to cytotoxicity of the superficial cell layer of the urothelium (23). As discussed above, it appears that the anion of the sodium salt, such as saccharin or ascorbate, does not directly produce the urothelial toxicity. The structure of the anion is not changed by the other variations that occur in the urinary milieu (29), such as pH, and there is no evidence that these anions, such as saccharin, bind to a cell surface receptor (34). Since sodium salts, such as ascorbate and chloride, are essential ingredients of the diet or intermediates of cell metabolism (e.g. glutamate, bicarbonate, aspartate, succinate), it is not surprising that they are handled metabolically somewhat differently than saccharin. Saccharin is not metabolized (30), and most of it is excreted in the urine within 24 h of administration (30). Concentration in the urine can reach levels as high as 200–300 mM following the administration of various saccharin salts at 5 or 7.5% of the diet. In contrast, administration of sodium ascorbate at similar doses in the diet produces nearly one to two orders of magnitude lower concentration in the urine of the corresponding anion, ascorbate (as ascorbate plus dehydroascorbate). These findings also suggest that the cytotoxicity of the urothelium following administration of high doses of these sodium salts occur by an indirect mechanism.

Nearly two decades ago Arnold et al. (8) observed that a precipitate formed in the urine following administration of high doses of sodium saccharin, and this was corroborated by observations by West and Jackson (36). We have also observed this precipitate in the urine of rats fed high doses of sodium saccharin (37,38), and in the urine of rats fed various other sodium salts (38,67) that have been found to produce urothelial proliferation.

Formation of this precipitate appears to be necessary for the urothelial cytotoxicity, hyperplasia and tumorigenicity of these sodium salts. Any treatments that inhibit the formation of this precipitate inhibit the induction of the toxicity, proliferative and tumorigenic effects. For example, one of the necessary conditions for the formation of this precipitate is a pH of ~6.5 or greater (37,38,67). If the rat is treated in such a way that it produces acidic urine, no precipitate forms and there is no toxicity, regeneration, or tumor formation (1719,38,49,67). Acidification can be produced either by administration of the parent acid, such as acid saccharin (17,33) or ascorbic acid (39), or by the sodium salt co-administered with high doses of ammonium chloride (38,49,67). Administration of the sodium salt in AIN-76A, a semi-synthetic diet with casein as the protein source, also results in acidic urine (49) and inhibition of the urinary and urothelial effects of sodium salts (19,70).

An apparent exception to the tumorigenicity of the sodium salts is sodium hippurate (71). Administration of this salt at high doses in the diet to male rats does not produce proliferation or tumor formation. However, the administration of high doses of sodium hippurate leads to a urine pH of ~6.3 or less, which is not conducive to the formation of the precipitate.

The formation of the precipitate is also dose dependent. The precipitate is consistently present at high doses of sodium saccharin, occasionally present at a dose of 3% of the diet and absent at a dose of 1%. The tumorigenic effects of sodium saccharin on the urothelium show a similar dose response with a significant increase in bladder tumors occurring at doses >=3% (9).

The present studies also demonstrate that calcium phosphate precipitate is cytotoxic when added to the medium of urothelial cells in culture. Calcium phosphate has long been known to be cytotoxic to epithelial cells (72), and the observations with urothelial cells adds to the long list of other epithelia that respond to this precipitate. In contrast to epithelial cells, calcium phosphate precipitate is not cytotoxic to mesenchymal cells, such as fibroblasts, but rather has been used as an agent to transfect DNA into such cells (72). The present studies demonstrate that the precipitate is formed soon after sodium saccharin administration begins, and that it may form high in the urinary tract, being present in the renal pelvis as well as the ureter and the urinary bladder.

The major components of the precipitate appear to be calcium and phosphate, present as Ca3(PO4)2, and the major organic component appears to be sulfated acid mucopolysaccharides, principally chondroitin sulfate. Significant amounts of urea are also present. Small amounts of saccharin (in rats fed sodium saccharin) and protein are also present along with small amounts of silica. Whether magnesium is a constituent of the precipitate or only a contaminant representing the magnesium ammonium phosphate in crystals also present in the urine could not be definitely ascertained. However, the more we were able to eliminate crystals from the precipitate, the less magnesium that was present in the material.

The composition of the precipitate appears to vary considerably between animals and even within the same animal at different times. Although calcium and phosphate, urea, and mucopolysaccharide were always present, the proportion of each varied considerably. Likewise, the other components varied widely between the rats. The analyses that were performed both qualitatively and quantitatively, using X-ray energy dispersive spectroscopy, infrared spectroscopy and chemical analyses, provided means to establish the general composition of the precipitate.

It is clear that protein is present in the precipitate, but it is present only in small amounts. The role that the protein plays in this precipitate is unclear. We have suggested (34,37) that it might principally be acting as a nidus for the formation of the urinary precipitate, a common process in the formation of urinary solids such as precipitate, crystals and calculi (73).

The presence of large amounts of protein in rat urine appears to explain why the rat is susceptible to the formation of this precipitate as well as the proliferative and tumorigenic activity of these sodium salts, and also helps to explain the difference in response between male and female rats. Although the urine of the male rat has considerably more protein than the urine of the female rat, the female rat does have a large amount of protein, mostly albumin. The difference between the sexes is due to the presence of large amounts of {alpha}2u-globulin in the urine of male rats (74). It is probable that this contributes significantly to the biological effects in the urinary tract following administration of these sodium salts. For example, administration of sodium saccharin (75,76) or sodium ascorbate (76) to the NBR male rat, which does not excrete {alpha}2u-globulin in large quantities in the urine, produces significantly less proliferative effect than does administration to Fischer or Sprague–Dawley rats, which have large amounts of {alpha}2u-globulin. The response in the NBR male rat to these salts is similar to the response of female rats of other strains.

These proteins are present in normal rat urine, and yet calcium phosphate does not usually precipitate in the urine of control animals. The changes that occur in the urine of rats treated with high doses of these sodium salts that leads to formation of this precipitate are unclear. The anions of these sodium salts, such as saccharin or ascorbate, are known to non-covalently associate with proteins, such as albumin and to an even greater extent to {alpha}2u-globulin, which may alter their structure in a way that leads to formation of this precipitation. The role of mucopolysaccharides in this process is also unclear, but could possibly be related to the interaction of calcium and phosphate.

The role of the extremely high osmolality of rat urine in the formation of the precipitate is also unclear. Rat urine, like that of other rodents, is very concentrated, especially in contrast to the urine of primates, such as humans (77). Human urinary osmolality is usually in the range of 50–500 mosm/l. Theoretically it has been calculated that osmolality in human urine may reach levels as high as 1000–1200 mosm/l. Rat urine routinely has an osmolality above 1000 mosm/l, even >2000 mosm/l. This increased osmolality is due partly to the significantly greater concentrations of urea in rat urine than in human urine (77). Increased urine volume with distension of the bladder has also been suggested as contributing to the tumorigenicity of the sodium salts (78). In contrast, increased water and total liquid consumption are related to a decreased risk of bladder cancer in men (79).

It has been suggested that high sodium concentrations along with pH by itself are adequate to produce the proliferative response of the sodium salts (80). However, high urinary pH by itself, clearly, is not adequate to produce these effects. For example, the feeding of a commonly used rodent chow in Europe, Altromin 1321 (Altromin GmbH and Co., Germany), routinely produces an urinary pH of 8.0 or higher, and yet the bladder epithelium of control animals fed this diet remains normal (81). Similarly, high sodium levels are produced at similar levels in male and female rats fed high doses of sodium saccharin, and yet the response of the urothelium is clearly greater in the male rat (see Ref. 82 and present studies). Even higher sodium concentrations can be achieved in mice fed high doses of these sodium salts, and yet there is no toxic or proliferative response in their urothelium (83).

An explanation is necessary for the lack of effect in the urothelium of mice fed high doses of these sodium salts. When sodium saccharin is fed at concentrations of 5 or 7.5% of the diet, even higher concentrations of sodium and saccharin occur in the urine of mice than in rat (83). Also, the mouse has similar high urinary protein concentrations, and the mouse has a protein analogous to {alpha}2u-globulin, mouse urinary protein (MUP) (84). MUP associates with anions such as saccharin in a manner and to an extent similar to that of {alpha}2u-globulin (37,85). Nevertheless, we have not observed formation of the urinary precipitate (unpublished observations), and there has been no evidence of increased proliferation or tumorigenicity in mice administered sodium saccharin (86).

The mouse has urinary concentrations of calcium, phosphate and magnesium that are significantly lower than in the rat (77). This is particularly true for calcium, where the concentration is as much as 10–20 times greater in the rat than in the mouse. Since the solubility product for calcium phosphate is a multiplicative rather than an additive function of the concentration of these components, it is likely that the large differences in concentration is at least a partial explanation for lack of precipitate formation in the mouse. It also points out an additional correlation between the presence or absence of the formation of this precipitate and the presence or absence of a consequent toxic or proliferative response.

{alpha}2u-Globulin has been associated with renal carcinogenesis for a variety of compounds that reversibly bind to the protein, are absorbed into the proximal tubules, are essentially undigested and accumulate in these tubular cells, and lead to tubular cell death and consequent regeneration (87). There is some indication that {alpha}2u-globulin might also contribute to the aging nephropathy common in male rats. It does not appear that saccharin associates with the {alpha}2u-globulin in the same way that rat renal carcinogens, such as D-limonene (L.Lehman-McKeeman, personal communication). Of interest has been the repeated observation with the sodium salts that long term administration at high doses tends to inhibit the formation of the aging nephropathy that occurs in male rats over time (69). This inhibitory effect can be at least partially reversed by co-administration with ammonium chloride (48), suggesting that the interaction of these anions with {alpha}2u-globulin occurs within the renal parenchyma and not initially within the renal pelvis.

In summary, a precipitate forms in the urine of rats administered high doses of sodium salts such as saccharin and ascorbate, and it appears to be an essential determinant in the toxicity of the urothelium in rats fed high doses of these compounds with consequent regeneration and tumor formation. The precipitate is composed principally of calcium phosphate, mucopolysaccharides and urea, with protein, anions (such as saccharin), and silica also being present possibly along with magnesium. Precipitated calcium phosphate is cytotoxic to urothelial cells, as is the calcium phosphate-containing urinary precipitate that forms in these rats. The precipitate appears to act directly on the superficial cells without being incorporated into the epithelium. The conditions necessary for the formation of the precipitate such as, high urinary pH (>=6.5), high urinary protein and osmolality, large amounts of urinary mucopolysaccharides, and adequate levels of calcium and phosphate (and possibly magnesium) suggest that the rat is the only sensitive species to the urothelial effects of feeding high doses of these sodium salts. The male rat is more susceptible than the female, and for reasons discussed above, mice and primates are not susceptible to the effects of high doses of these sodium salts because they do not produce the necessary conditions for the formation of this precipitate. The data presented, in addition to other observations, suggest that the tumorigenicity following administration of high doses of these sodium salts is a high dose, rat specific phenomenon.


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Table II. Cytotoxicity of calcium phosphate precipitate to rat bladder epithelial cells
 

    Acknowledgments
 
We gratefully acknowledge the assistance of Dr Angela Mann, Steve Eklund, Margaret St John, Traci Anderson, Linda Johnson, Cindi Lear and Barbara Mattson with the performance of these studies, and Denise Miller and Michelle Moore with the preparation of the manuscript. The research was supported in part by USPHS grants CA32513 and CA36727 from the National Cancer Institute and a grant from the International Life Sciences Institute. S.M.C. is the Wall-Havlik Professor of Oncology.


    Notes
 
4 Present address: 8204 Londonberry Road, Nashville, TN 37221, USA Back

5 To whom correspondence should be addressed Email: scohen{at}unmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 22, 1999; revised November 17, 1999; accepted November 29, 1999.