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Some changes in gastro-intestinal metabolism and in the urine and bladders of rats in response to sodium saccharin ingestion
Fd Chem. Toxic. Vol. 23, No. 4/5, pp. 457-463, 1985
0278-6915/85 $3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SOME C H A N G E S IN G A S T R O - I N T E S T I N A L M E T A B O L I S M A N D IN THE U R I N E A N D B L A D D E R S OF RATS IN RESPONSE TO S O D I U M S A C C H A R I N I N G E S T I O N R. L. ANDERSON The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 39175, Cincinnati, OH 45247, USA
Abstract--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.
NaS ingestion induces many changes in urine composition, including an increase in the urea:ammonia ratio without changing the total urinary nitrogen and an acid shift in pH (Anderson, 1979). The NaS-associated polyuria is associated with a compensatory decrease in urine osmolality (Schoenig & Anderson, 1985). NaS ingestion also increases urinary SO~-, C1- and citrate but does not alter creatinine (P. Werness, personal communication, 1980) or lactate excretion. It has recently been observed that ingestion of NaS at 5 or 7.5~o in the diet induces an increase in bladder-tissue concentrations (pmol/g dry tissue) of K, Mg and Zn in male, but not in female, rats. In males at these dietary levels no significant change in tissue Ca was observed, and a significant increase in bladder-tissue Na occurred only at the 7.5% dietary level. The available information on NaS suggested that the primary site of activity was in the gastro-intestinal (GI) tract and that the urine and bladder changes were probably a consequence of these alterations in GI metabolism. This report will focus on experiments designed to determine if the urine and bladder changes noted in rats ingesting NaS are a consequence of its effects of GI metabolism.
Introduction Several authors have reported that ingestion of sodium saccharin (NaS) is associated with a dosedependent increase in caecal volume and stool hydration (Anderson, 1979 & 1983; Lawson & Hertzog, 1981; Schoenig & Anderson, 1985; Sims & Renwick, 1983). NaS has also been associated with a dosedependent, rapidly induced and sustained polydypsia and polyurea (Anderson, 1979; Renwick & Sims, 1983; Schoenig, Goldenthal, Geil et al. 1985). The increased stool hydration has been attributed to the high stool concentration of NaS and the voiding of hygroscopic polysaccharides that may be derived from the diet or synthesized by an intestinal microorganism(s) (Anderson, 1983). At least part of the caecal enlargement has been attributed to an increased amount of ingested protein being transported to the lower bowel, probably because of NaS-induced inhibition of pepsin (Lok, Iverson & Clayson, 1982) and trypsin (Sims & Renwick, 1985). NaS ingestion has also been shown to alter the disposition of dietary minerals by increasing the faecal excretion of Na and K while decreasing faecal Mg and P. In contrast, the urine contains increased amounts of Na, Ca, Mg and P (Anderson, 1979; Schoenig et al. 1985; Schoenig & Anderson, 1985). In spite of these changes in the site and type of metabolism of ingested nutrients, ingestion of NaS does not significantly alter the net absorption (ingested minus stool) of dietary non-saccharin nitrogen or lipid, and results in only a small increase in total stool ash (Anderson, 1983). NaS does cause a dosedependent decrease in feed efficiency (weight gain/100g diet consumed; Anderson, 1979) which can be largely accounted for by the dose-dependent increase in stool polysaccharide (Anderson, 1983). ERF = epithelial-rich fraction; GI tract = gastro-intestinal tract; NaS = sodium saccharin; VFA = volatile fatty acid.
Abbreviations:
Experimental Animals. Weanling male and female Sprague-Dawley-derived (CR-CD, 21-days-old) and Fischer (CDF, 28-days-old) rats were obtained from Charles River Breeding Laboratories (Wilmington, MA). Weanling (21-days-old) male Sprague-Dawley rats were obtained from ARS Laboratory Animal Supply (Madison, WI). All rats were housed individually in stainless-steel, suspended wire-mesh-floor cages and provided a d lib. with distilled water and ground Purina Lab Chow (Ralston Purina Co., St Louis, MO). The rats were observed for signs of disease for at least 3 days before they were placed on
457
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R.L. ANDERSON
study. The room was maintained at 72 ___5°F and 50 + 20% relative humidity with a 12-hr alternating light/dark cycle. Diets. Sodium saccharin (Lot 1648, Sherwin Williams Co., Cincinnati, OH) was added to either ground Purina Chow at 5.0 or 7.5% or to a purified diet, AIN-76A (Teklad Test Diets, Madison, WI), at 7.5%. The appropriate control diets were made isocaloric by the addition of either 5.0 or 7.5% cellulose. Feeding studies. Following acclimatization, the rats were randomly allocated to groups of 5 on the basis of initial body weights. All animals had free access to feed and distilled water. Feed and water consumption were determined twice a week, and body weights were measured weekly. At a specified time during the studies, 24-hr urine samples were collected from animals fitted with faecal collector cups. Daily records of food and water consumption and body weight were maintained during the urine-collection periods. Infusion studies. Young adult male CR-CD rats (200-250g body weight) were anaesthetized with Nembutal (40mg/kg, ip) and restrained. A small incision was made through the skin over the right jugular vein and the vein was exposed by blunt dissection. Two sutures of 4-0 silk were tied around the vein: tightly at the cranial tie and loosely at the caudal tie. A small incision was made only part of the way through the jugular vein and a silicon rubber cannula (0.020in. I.D. x0.037 in O.D., DowCorning Silastic; Dow-Corning Corp., Midland, MI) with a double-bevel cut on the end was placed in the vein and tied securely with the caudal suture. The cannula was secured with several subcutaneous ties and passed beneath the skin along the back and brought to the surface near the base of the tail. The incision was dosed with 4-0 silk sutures. The rats were placed in wire-mesh restrainers designed for the collection of urine. Using Harvard Model 975 infusion pumps, each animal was infused daily with 2.7 or 4.0 g NaS/kg body weight/day. Control animals were given an equivalent daily Na load as saline. The infusion rate was 0.01 ml/min or 14.4ml/day. Food and water consumption and urine volumes were measured daily. The rats were infused until the cannula was no longer functional or for 5 days, whichever occurred first. Enzyme assays. The ability of NaS to inhibit the hydrolysis of starch was determined using a commercial preparation of porcine ~¢-amylase (lot no. 101848, Calbiochem-Behring Corp., La Jolla, CA). Sucrase and isomaltase were prepared as outlined by Conklin, Yamashiro & Gray (1975) from the small intestines of rats. The tissue preparation used was the supernatant of the 100,000-g centrifugation after tissue autolysis. The tissue was prepared from rats ingesting a purified diet (AIN TM 76) for sucrase activity measurements and from rats fed laboratory chow for isomaltase activity. All enzymes were assayed in 0.02 M-phosphate buffer at pH 6.0. Soluble starch (Matheson, Coleman & Bell, Norwood, OH) hydrolysis was measured by determination of the loss of iodine binding. Sucrase and isomaltase activities were determined by measuring the rate of glucose production using a glucose analyser (Beckman Glucose Analyzer No. 2, Beck-
man Instruments Inc., Fullerton, CA). Sucrose activity was measured using 28 raM-sucrose as substrate and isomaltase activity was determined using corn solids (Fro-Dex, O, American Maize Co., Hammond, IN) as substrate. The addition of maximal levels of NaS (100mg/ml) after hydrolysis did not alter the detection of products in any of the assays. An aqueous extract of chow was prepared by homogenization of laboratory chow in water (1 g/10ml) and centrifugation at 1150 RCF for 10 min. Caecal incubations. For determination of transport of volatile fatty acids (VFAs) and lactate from caeca in vitro, ligated caeca plus their contents from 3 control rats and 3 rats that had been fed NaS for 28 days were incubated in 15 ml Krebs-Ringer phosphate buffer (pH 7.4) at 37°C in a shaking water bath. The buffer was changed twice after a 1-hr incubation period and the third medium was incubated for 2 hr. Each medium sample was brought to pH 10 and frozen. At the end of the incubation period the caecal contents were brought to pH 10 and frozen. Lactate and VFA analyses were conducted on each sample. Analytical determinations. The concentration of VFAs in the caecal contents and media were determined by gas chromatography using a standard analytical method (the author will provide details on request). Lactic-acid concentrations were measured using a commercial lactate dehydrogenase method (Sigma Chemical Co.). All urine and tissue samples were wet-ashed using a perchloric acid/nitric acid procedure. Mineral concentrations were measured by flame ionization using a Perkin-Elmer 603 atomic absorption spectrophotometer. The concentration of saccharin in the urine was measured by high-pressure liquid chromatography. Saccharin was detected by UV absorption at 245 nm. Results Analyses of faeces demonstrated that NaS ingestion causes a dose-dependent increase in the faecal content of a soluble polysaccharide (Anderson, 1983). It was subsequently shown that a Streptococcus organism isolated from the caecum synthesized an extracellular polysaccharide when grown in a sugarsupplemented medium (Shibata, Goldstein & Kirkland, 1983). Polysaccharide synthesis by this Streptococcus was not influenced by the presence of NaS in the medium (up to 100 mg/ml) whether the medium contained excess sugar (polysaccharide production) or not (no polysaccharide synthesis) (J. J. Kirkland, personal communication, 1982). These findings suggested that ingestion of NaS was making increased amounts of dietary carbohydrate available to the microflora of the lower GI tract. To test this hypothesis the effect of NaS on the activity of the enzymes associated with dietary carbohydrate hydrolysis (pancreatic amylase and intestinal sucrase and isomaltase) was ascertained. NaS caused a concentration-dependent inhibition of hydrolysis of starch by amylase (Fig. 1) and of sucrose (Fig. 2a) and corn-syrup solids (Fig. 2b) by an intestinal fraction containing sucrase and isomaltase activity. Figure 3 shows that NaS inhibits glucose production from an aqueous extract of chow by the intestinal
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Fig. l. Inhibition by sodium saccharin of starch hydrolysis by porcine pancreatic amylase. Each value is the mean of at least three determinations.
Fig. 3. Inhibition by sodium saccharin o f glucose production from an aqueous extract of laboratory chow by a small-intestinal fraction from rats. Each value is the mean o f three assays and range bars indicate the SEM.
fraction and therefore that the inhibition by NaS of hydrolysis of dietary carbohydrate is applicable to rat feeding studies., The demonstration that NaS can inhibit the enzymes responsible for dietary carbohydrate hydrolysis when coupled with its previously reported inhibition of the enzymes associated with hydrolysis of dietary protein (see Introduction) suggested a possil~le causality between the NaS dose-dependent enzyme inhibition and polydypsia. It was hypothesized that polydypsia was induced in order to provide sufficient intestinal fluid for lower bowel fermentation of the increased dietary components made available to the lower GI tract. This hypothesis was tested by ascertaining whether NaS ingestion altered the dry mass of the caecal contents and by determining whether systemic NaS (given iv) altered water consumption. Table 1 shows that while ingestion of NaS at 7.5% in the diet or 4% in drinking-water caused a doubling of water consumption and a greater than two-fold increase in the relative mass (g/kg body weight) of the caecal contents, it caused only a small increase in the water content of the caecal contents. In contrast, rats infused iv with 2.7 or 4.0 g NaS/kg body weight/day (equivalent to the systemic NaS load induced by 7.5 or 11.25% dietary NaS) for 5 days did not consume significantly more water than controls. Thus it can be concluded that ingestion of
NaS induces polydypsia in order to provide water for the hydration of the caecal contents; there is no such response to NaS given systematically. In order to ascertain whether the increased caecal fermentation that results from NaS ingestion alters the end-products of fermentation that are delivered to the host the lactate and VFA concentrations in caecal contents and their delivery to a medium during incubation in vitro were compared in caeca from control rats and rats given a diet containing 5% NaS. NaS increased the caecal concentration of lactate and decreased the caecal concentration of VFAs. The transport into the medium of these end-products of fermentation reflected the caecal concentrations (Fig. 4a, b). Comparison of the concentration of the individual VFAs in the medium and the caecal contents showed that NaS did not alter the acetate level but decreased the propionate (C3) and especially the butyrate (C4) and the valerate (C5) levels (Fig. 5). Filtrates (0.2-#m filter) of the caecal contents and media following incubation showed no mutagenic activity in Salmonella tester strains TA98 and TA100 with or without microsomal activation (E. D. Thompson, personal communication, 1982). There were no significant differences between male and female rats in any of the above observations of the effects of NaS on the GI tract. The above findings led to the hypothesis that the
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460
R. L. ANDERSON Table 1. Water consumption and caecal parameters in male rats given sodium saccharin in the diet or by iv infusion
Treatment Control: NaS:
7.5% dietary cellulose 7.5% in diet 4% in drinking-water 2.7 g/kg body weight/day, iv 4.0 g/kg body weight/day, iv 0.9% (w/v) aqueous solution, iv
NaCI:
Water consumption (g/g diet consumed)
Relative caecal weight g/kg body weight)
Caecal water (g/ 100 g of caecal contents)
pH of caecal contents
1.7 + 0.04 3.3 + 0.2 3.0 -I- 0.4 2.1 ___0.1 2.0 + 0.1 1.8 __.0.1
29 + 2 62 + 3 75 + 24 15 ___2 ND 18 + 1
78 83 84 ND ND ND
5.9 5.9 6.0 ND ND ND
NaS = Sodium saccharin ND = Not determined Each value is the mean of five or six determinations ( + S E M for water consumption and relative caecal weight). NaS was given in the diet or in drinking-water for 28 days, and iv infusion was carried out for 5 days. Infusion with 2.7 or 4.0 g NaS/kg body weight/day provided a systemic load of NaS equivalent to that induced by 7.5 or 11.5% NaS given in the diet.
polyuria and increased concentration of ions (CI-, PO34- , SO42-, Ca 2÷, Mg 2+ and Na +) in the urine that have been observed in rats fed NaS (Anderson, 1979; Schoenig & Anderson, 1985; Schoenig et aL 1985) result from increased caecal absorption of these ions and their passive transport into the urine and are not a specific response to renal clearance of NaS. To test this hypothesis the effect of iv-administered NaS and NaC1 on plasma concentrations and urinary excretion of selected minerals was ascertained. Table 2 shows that the administration iv of 11 mmol NaS/kg o
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body weight/day (the systemic load associated with a dietary level of 7.5% NaS) for 5 days results in plasma concentrations and urinary excretions (#mol/g diet ingested) of Ca, Mg, Na and P that are similar to those in rats given an equivalent iv dose of NaCL These values are not greatly different from those recorded in non-infused controls except for a higher urinary excretion of Na, undoubtedly due to the high dose being given. More than 95% of the infused saccharin was excreted in the urine. Thus, it can be concluded that the increased urinary excretion of ions that has been noted in rats ingesting NaS is due to increased absorption of these ions from the GI tract and is not a consequence of inhibition of their reabsorption during the renal clearance of NaS. Elsewhere in this issue it is reported that ingestion of NaS is associated with an increase in the relative bladder mass (g/kg body weight) that correlates with NaS-induced polyuria (Schoenig & Anderson, 1985). This work also shows that increased bladder mass was accompanied by an increase in the concentration (#moi/g dry tissue) of several cations in the bladder tissues of male rats but not of female rats (Schoenig & Anderson, 1985). The effect of 5 days of infusion of NaS or NaCI (c. 11 mmol/kg body weight/day) on bladder mass and cation concentrations in male rats
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Fig. 4. Release o f (a) volatile fatty acids ( V F A s ) a n d (b) lactate into K r e b s - R i n g e r buffer d u r i n g the in vitro incub a t i o n o f caeca f r o m m a l e c o n t r o l rats ( x ) a n d m a l e rats fed s o d i u m saccharin a t 5% in the diet for 28 d a y s ( 0 ) . A l s o s h o w n are the caecal contents o f V F A a n d lactate in c o n t r o l ( [ ] ) a n d saccharin-fed ( B ) rats after i n c u b a t i o n . Values are m e a n s o f three d e t e r m i n a t i o n s .
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Gastro-intestinal effects of saccharin in rats
461
Table 2. Mineral levels in plasma and urine o f male rats infused iv with sodium saccharin o r s o d i u m chloride Plasma* o r urinet levels o f Treatment
Ca
Mg
Masma 2.28 _+ 0.05 0.79 + 0.05 2.30 _+ 0.04 0.74 _+ 0.03
Saline Sodium saccharin Saline Sodium saccharin
4.2 + 0.2 4.5 __.0.3
Urine 23 _ 2 20 _ 2
Na
P
154 _ 6 165 _ 4
4.26 _ 0.22 3.56 _ 0.23
302 + 28 296 +_ 32
37 _+ 4 36 _+ 6
*Values are for m e a n concentrations (raM) __. S E M for six samples taken after 5 days o f iv infusion. "tValues are for m e a n levels (/tmol/g diet consumed) _+ S E M for five daily determinations on six rats/treatment. Sodium saccharin and s o d i u m chloride were infused at 11 m m o l / k g body weight/day at a rate o f 14.4 ml/rat/day.
was measured to ascertain whether the responses noted in rats ingesting NaS were a specific response to high systemic levels of NaS or were a response to polyuria. There was no decrease in water consumption (Table 1) by rats infused with NaS or NaCI at a rate of 14.4ml/day. Therefore, this treatment does cause polyuria, approximately doubling urinary volume in comparison with non-infused controls. Figure 6 shows that dosing iv with either NaS or
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Sodium soechorin (g/kg/doy) Fig. 6. Effect on relative dry bladder mass (mg/kg body weight) of giving sodium saccharin (m) in the diet (7.5~) or by iv infusion (2.7 g/kg body weight/day) to male rats in comparison with that in control rats (ll) given basal diet or iv infusions of sodium chloride (0.9~ aqueous solution). Each rat weighed c. 250 g; sodium saccharin was given in the diet for 3 wk and iv infusion of sodium saccharin or sodium chloride was carried out for 5 days using an infusion volume of 14.4ml/rat/day. The systemic load of sodium saccharin was approximately equivalent for the orally and iv-dosed rats (c. 11 mol/kg body weight/day). Values are means for 5 rats and range bars indicate the SEM.
NaC1 for only 5 days results in a greater increase in relative dry bladder mass (mg/kg body weight) than does 3 wk of ingestion of a diet containing 7.5~ NaS (a concentration that results in a systemic NaS load of c. 11 mmol/kg body weight/day). The effects of these iv doses of NaS and NaCI on ion concentration in bladder tissue ~ m o l / g dry tissue) in comparison with the effect of 21 days of ingesting 7.5~ NaS are summarized in Fig. 7. Administration iv of either NaS or NaCI for only 5 days resulted in greater ion accumulation in bladder tissue than did 21 days of 7.5~ dietary NaS. This work allows three conclusions: (1) ion accumulation in the bladder is not dependent upon increased systemic loads of the ions involved (see Table 2); (2) mineral accumulation in the bladder is not dependent upon high urinary levels of NaS; (3) increased bladder mass in response to polyuria is a sufficient stimulus to induce ion accumulation in bladder tissue ( N a C 1 ) NaS). Since all of the bladder tumours that have been reported in rats ingesting NaS in chronic studies have occurred in the bladder epithelium (Arnold, Moodie, Grice et al. 1980; Schoenig et al. 1985; Taylor, Weinberger & Friedman, 1980; Tisdel, Nees, Harris & Derse, 1974), the site of the mineral accumulation in the bladders of male rats ingesting NaS at 7.5~o in the diet was ascertained by preparing an epithelial rich fraction (ERF) by scraping the luminal surface of everted bladders. The mineral concentrations in the ERF and total bladders from control rats and those ingesting 7.5~ NaS are shown in Fig. 8. The ERF fractions from the rats ingesting NaS was 9.5 _+0.4~o (mean _+ SEM) of the tissue total dry mass (ERF plus residue) compared with 7.9 _+ 1.3~o for the controls. In the control samples the concentration of each of the minerals determined except Na and Zn was higher in the ERF than in the whole bladder tissue. The mineral concentrations in the ERF from the rats fed NaS were higher than those in the controls. In fact while the ERF constituted only c. 10~ of the tissue dry mass, it contained the majority of the increased minerals associated with NaS ingestion. Discussion
The major purpose of the work described in this report was to ascertain whether the effects on the
462
R . L . ANDERSON
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Fig. 7. Mineral concentrations in the bladders of male rats given sodium saccharin in the diet (7.5~) for 3 wk ([]) or by iv infusion (2.7 g/kg body weight/day) for 5 days (~l) in comparison with those in control rats fed the basal diet (ll) or infused iv for 5 days with a 0.9~ aqueous solution of sodium chloride ([]). Each rat weighed c. 250 g. The systemic load of sodium saccharin was approximately equivalent for the orally and iv-dosed rats (c. 11 mmol/kg body weight/day). Each value is the mean for five rats (feeding study) or four rats (iv study) and range bars indicate the SEM. urine and bladder that have been observed in rats fed NaS in chronic studies could be shown to be a consequence of changes in dietary nutrient metabolism in the GI tract induced by the NaS. The results reported here are clearly consistent with the concept that the primary site of NaS effects in the rat is in the GI tract and that the alterations in the urine and bladder tissue are secondary responses. However, more important is the examination of the effects observed for their possible contribution to epithelial tumorigenesis in the rat bladder. An increase in the incidence of urinary bladder tumours has been observed in male rats fed NaS at ) 3 ~ in the diet and in two-generation studies the tumour incidence was greater in F, than in F0 rats (Arnold et No
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al. 1980; Schoenig et al. 1985; Taylor et al. 1980; Tisdel et al. 1974). In order for a condition to be
considered as a potential 'causative' factor in bladder tumorigenesis, that factor should show a response to NaS similar to that of the tumorigenesis. In this study no differences were observed between male and female rats in the GI effects of NaS, and neither has a greater response been observed in Ft than in F 0 rats (data not shown). Therefore the changes described in this report are not sufficient to be considered as the 'causal' factor(s) in bladder tumorigenesis. This does not mean, however, that the changes noted are not 'necessary' conditions for tumorigenesis. The fact that dietary NaS produces a male-specific mineral accumulation in the bladder fraction contain-
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Fig. 8. Mineral concentrations in the 'epithelial-rich fraction' (ERF) of the bladders of control rats (m) and rats fed sodium saccharin at 7.5~o in the diet for 3 wk (m), in comparison with those in the whole bladder tissue (TB) from comparable rats. The ERF was prepared by scraping the bladder surface with a glass slide. The ERF values are means of three determinations on ERF pooled from the bladders of three rats and the TB values are the means of 5 samples. All samples were obtained from c. 7-wk-old male CR (CD) rats.
Gastro-intestinal effects of saccharin in rats ing the epithelium is the most pertinent observation reported, in that this response occurs in the same sex and at the same organ site in which tumorigenicity is noted. The fact that the male-specific mineral accumulation in the bladder is rapidly induced by the ingestion of high doses of NaS and that this response is no more pronounced in F1- than in F0-generation animals would certainly preclude the conclusion that mineral accumulation in the bladder is the 'cause' of bladder tumours in rats fed high doses of NaS in chronic studies. Furthermore, even if mineral accumulation in the bladder is necessary for tumorigenesis, the observation that iv-administered NaCI is as effective as iv-administered NaS in inducing mineral accumulation in the bladder would suggest that any c o m p o u n d that causes increased water absorption and polyuria should be able to induce bladder tumors in male rats. In this regard, it should be noted that analysis of the results o f a recent bioassay of NaS has revealed a strong correlation between polyuria early in life and the subsequent development of bladder turnouts a m o n g rats fed NaS at 7.5% in the diet (Schoenig et al. 1985). In summary, the studies reported show that NaSassociated changes in urine composition, increased bladder-tissue mass, and the male-specific mineral accumulation in bladder tissue in rats are consequences of the changes in GI metabolism that are associated with NaS-induced inhibition of the enzymes that digest dietary proteins and carbohydrates. While these findings have not identified a causal factor(s) for NaS-associated bladder tumorigenesis in rats, they do provide a mechanism for comparison between species of the effects of NaS at its primary site of activity.
REFERENCES
Anderson R. L. (1979). Response of male rats to sodium saccharin ingestion: urine composition and mineral balance. Fd Cosmet. Toxicol. 17, 195.
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Anderson R. L. (1983). Effect of saccharin ingestion on stool composition in relation to caecal enlargement and increased stool hydration. Fd Chem. Toxic. 21, 255. Arnold D. L., Moodie C. A., Grice H. C., Charbonneau S. M., Stavric B., Collins B. T., McGuire P. F., Zawidzka Z. Z. & Munro I. C. (1980). Long-term toxicity of ortho-toluenesulfonamide and sodium saccharin in the rat. Toxic. appl. Pharmac. 52, 113. Conklin K. A., Yamashiro K. M. & Gray G. M. (1975). Human intestinal sucrase-isomaltase. Identification of free sucrase and isomaltase and cleavage of the hybrid into active subunits. J. biol. Chem. 250, 5735. Lawson T. A. & Hertzog P. J. (1981). The failure of chronically administered saccharin to stimulate bladder epithelial DNA synthesis in F 0 rats. Cancer Lett. 11, 221. Lok E., Iverson F. & Clayson D. B. (1982). The inhibition of urease and proteases by sodium saccharin. Cancer Lett. 16, 163. Renwick A. G. & Sims J. (1983). Distension of the urinary bladder in rats fed saccharin containing diet. Cancer Lett. 18, 63. Schoenig G. P. & Anderson R. L. (1985). The effects of high dietary levels of sodium saccharin on mineral and water balance and related parameters in rats. Fd Chem. Toxic. 23, 465. Schoenig G. P., Goldenthal E. I., Geil R. G., Frith C. H., Richter W. R. & Carlborg F. W. (1985). Evaluation of the dose response and in utero exposure to saccharin in the rat. Fd Chem. Toxic. 23, 475. Shibata S., Goldstein I. J. & Kirkland J. J. (1983). Structure of a water-insoluble d-glucan isolated from a Streptococcus organism. Carb. Res. 120, 77. Sims J. & Renwick A. G. (1983). The effects of saccharin on the metabolism of dietary tryptophan to indole, a known carcinogen for the urinary bladder of the rat. Toxic. appl. Pharmac. 67, 132. Sims J. & Renwick A. G. (1985). The microbial metabolism of tryptophan in rats fed a diet containing 7.5% saccharin in a two-generation protocol. Fd Chem. Toxic. 23, 437. Taylor J. M., Weisberger M. A. & Friedman L. (1980). Chronic toxicity and carcinogenicity to the urinary bladder of sodium saccharin in the in utero-exposed rat. Toxic. appl. Pharmac. 54, 57. Tisdel M. A., Nees P. O., Harris D. L. & Derse P. H. (1974). Long-term feeding in rats. In Symposium Sweeteners. Edited by G. E. Inglett. p. 145. AVI Co., Westport, CT.
0278-6915/85 $3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SOME C H A N G E S IN G A S T R O - I N T E S T I N A L M E T A B O L I S M A N D IN THE U R I N E A N D B L A D D E R S OF RATS IN RESPONSE TO S O D I U M S A C C H A R I N I N G E S T I O N R. L. ANDERSON The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 39175, Cincinnati, OH 45247, USA
Abstract--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.
NaS ingestion induces many changes in urine composition, including an increase in the urea:ammonia ratio without changing the total urinary nitrogen and an acid shift in pH (Anderson, 1979). The NaS-associated polyuria is associated with a compensatory decrease in urine osmolality (Schoenig & Anderson, 1985). NaS ingestion also increases urinary SO~-, C1- and citrate but does not alter creatinine (P. Werness, personal communication, 1980) or lactate excretion. It has recently been observed that ingestion of NaS at 5 or 7.5~o in the diet induces an increase in bladder-tissue concentrations (pmol/g dry tissue) of K, Mg and Zn in male, but not in female, rats. In males at these dietary levels no significant change in tissue Ca was observed, and a significant increase in bladder-tissue Na occurred only at the 7.5% dietary level. The available information on NaS suggested that the primary site of activity was in the gastro-intestinal (GI) tract and that the urine and bladder changes were probably a consequence of these alterations in GI metabolism. This report will focus on experiments designed to determine if the urine and bladder changes noted in rats ingesting NaS are a consequence of its effects of GI metabolism.
Introduction Several authors have reported that ingestion of sodium saccharin (NaS) is associated with a dosedependent increase in caecal volume and stool hydration (Anderson, 1979 & 1983; Lawson & Hertzog, 1981; Schoenig & Anderson, 1985; Sims & Renwick, 1983). NaS has also been associated with a dosedependent, rapidly induced and sustained polydypsia and polyurea (Anderson, 1979; Renwick & Sims, 1983; Schoenig, Goldenthal, Geil et al. 1985). The increased stool hydration has been attributed to the high stool concentration of NaS and the voiding of hygroscopic polysaccharides that may be derived from the diet or synthesized by an intestinal microorganism(s) (Anderson, 1983). At least part of the caecal enlargement has been attributed to an increased amount of ingested protein being transported to the lower bowel, probably because of NaS-induced inhibition of pepsin (Lok, Iverson & Clayson, 1982) and trypsin (Sims & Renwick, 1985). NaS ingestion has also been shown to alter the disposition of dietary minerals by increasing the faecal excretion of Na and K while decreasing faecal Mg and P. In contrast, the urine contains increased amounts of Na, Ca, Mg and P (Anderson, 1979; Schoenig et al. 1985; Schoenig & Anderson, 1985). In spite of these changes in the site and type of metabolism of ingested nutrients, ingestion of NaS does not significantly alter the net absorption (ingested minus stool) of dietary non-saccharin nitrogen or lipid, and results in only a small increase in total stool ash (Anderson, 1983). NaS does cause a dosedependent decrease in feed efficiency (weight gain/100g diet consumed; Anderson, 1979) which can be largely accounted for by the dose-dependent increase in stool polysaccharide (Anderson, 1983). ERF = epithelial-rich fraction; GI tract = gastro-intestinal tract; NaS = sodium saccharin; VFA = volatile fatty acid.
Abbreviations:
Experimental Animals. Weanling male and female Sprague-Dawley-derived (CR-CD, 21-days-old) and Fischer (CDF, 28-days-old) rats were obtained from Charles River Breeding Laboratories (Wilmington, MA). Weanling (21-days-old) male Sprague-Dawley rats were obtained from ARS Laboratory Animal Supply (Madison, WI). All rats were housed individually in stainless-steel, suspended wire-mesh-floor cages and provided a d lib. with distilled water and ground Purina Lab Chow (Ralston Purina Co., St Louis, MO). The rats were observed for signs of disease for at least 3 days before they were placed on
457
458
R.L. ANDERSON
study. The room was maintained at 72 ___5°F and 50 + 20% relative humidity with a 12-hr alternating light/dark cycle. Diets. Sodium saccharin (Lot 1648, Sherwin Williams Co., Cincinnati, OH) was added to either ground Purina Chow at 5.0 or 7.5% or to a purified diet, AIN-76A (Teklad Test Diets, Madison, WI), at 7.5%. The appropriate control diets were made isocaloric by the addition of either 5.0 or 7.5% cellulose. Feeding studies. Following acclimatization, the rats were randomly allocated to groups of 5 on the basis of initial body weights. All animals had free access to feed and distilled water. Feed and water consumption were determined twice a week, and body weights were measured weekly. At a specified time during the studies, 24-hr urine samples were collected from animals fitted with faecal collector cups. Daily records of food and water consumption and body weight were maintained during the urine-collection periods. Infusion studies. Young adult male CR-CD rats (200-250g body weight) were anaesthetized with Nembutal (40mg/kg, ip) and restrained. A small incision was made through the skin over the right jugular vein and the vein was exposed by blunt dissection. Two sutures of 4-0 silk were tied around the vein: tightly at the cranial tie and loosely at the caudal tie. A small incision was made only part of the way through the jugular vein and a silicon rubber cannula (0.020in. I.D. x0.037 in O.D., DowCorning Silastic; Dow-Corning Corp., Midland, MI) with a double-bevel cut on the end was placed in the vein and tied securely with the caudal suture. The cannula was secured with several subcutaneous ties and passed beneath the skin along the back and brought to the surface near the base of the tail. The incision was dosed with 4-0 silk sutures. The rats were placed in wire-mesh restrainers designed for the collection of urine. Using Harvard Model 975 infusion pumps, each animal was infused daily with 2.7 or 4.0 g NaS/kg body weight/day. Control animals were given an equivalent daily Na load as saline. The infusion rate was 0.01 ml/min or 14.4ml/day. Food and water consumption and urine volumes were measured daily. The rats were infused until the cannula was no longer functional or for 5 days, whichever occurred first. Enzyme assays. The ability of NaS to inhibit the hydrolysis of starch was determined using a commercial preparation of porcine ~¢-amylase (lot no. 101848, Calbiochem-Behring Corp., La Jolla, CA). Sucrase and isomaltase were prepared as outlined by Conklin, Yamashiro & Gray (1975) from the small intestines of rats. The tissue preparation used was the supernatant of the 100,000-g centrifugation after tissue autolysis. The tissue was prepared from rats ingesting a purified diet (AIN TM 76) for sucrase activity measurements and from rats fed laboratory chow for isomaltase activity. All enzymes were assayed in 0.02 M-phosphate buffer at pH 6.0. Soluble starch (Matheson, Coleman & Bell, Norwood, OH) hydrolysis was measured by determination of the loss of iodine binding. Sucrase and isomaltase activities were determined by measuring the rate of glucose production using a glucose analyser (Beckman Glucose Analyzer No. 2, Beck-
man Instruments Inc., Fullerton, CA). Sucrose activity was measured using 28 raM-sucrose as substrate and isomaltase activity was determined using corn solids (Fro-Dex, O, American Maize Co., Hammond, IN) as substrate. The addition of maximal levels of NaS (100mg/ml) after hydrolysis did not alter the detection of products in any of the assays. An aqueous extract of chow was prepared by homogenization of laboratory chow in water (1 g/10ml) and centrifugation at 1150 RCF for 10 min. Caecal incubations. For determination of transport of volatile fatty acids (VFAs) and lactate from caeca in vitro, ligated caeca plus their contents from 3 control rats and 3 rats that had been fed NaS for 28 days were incubated in 15 ml Krebs-Ringer phosphate buffer (pH 7.4) at 37°C in a shaking water bath. The buffer was changed twice after a 1-hr incubation period and the third medium was incubated for 2 hr. Each medium sample was brought to pH 10 and frozen. At the end of the incubation period the caecal contents were brought to pH 10 and frozen. Lactate and VFA analyses were conducted on each sample. Analytical determinations. The concentration of VFAs in the caecal contents and media were determined by gas chromatography using a standard analytical method (the author will provide details on request). Lactic-acid concentrations were measured using a commercial lactate dehydrogenase method (Sigma Chemical Co.). All urine and tissue samples were wet-ashed using a perchloric acid/nitric acid procedure. Mineral concentrations were measured by flame ionization using a Perkin-Elmer 603 atomic absorption spectrophotometer. The concentration of saccharin in the urine was measured by high-pressure liquid chromatography. Saccharin was detected by UV absorption at 245 nm. Results Analyses of faeces demonstrated that NaS ingestion causes a dose-dependent increase in the faecal content of a soluble polysaccharide (Anderson, 1983). It was subsequently shown that a Streptococcus organism isolated from the caecum synthesized an extracellular polysaccharide when grown in a sugarsupplemented medium (Shibata, Goldstein & Kirkland, 1983). Polysaccharide synthesis by this Streptococcus was not influenced by the presence of NaS in the medium (up to 100 mg/ml) whether the medium contained excess sugar (polysaccharide production) or not (no polysaccharide synthesis) (J. J. Kirkland, personal communication, 1982). These findings suggested that ingestion of NaS was making increased amounts of dietary carbohydrate available to the microflora of the lower GI tract. To test this hypothesis the effect of NaS on the activity of the enzymes associated with dietary carbohydrate hydrolysis (pancreatic amylase and intestinal sucrase and isomaltase) was ascertained. NaS caused a concentration-dependent inhibition of hydrolysis of starch by amylase (Fig. 1) and of sucrose (Fig. 2a) and corn-syrup solids (Fig. 2b) by an intestinal fraction containing sucrase and isomaltase activity. Figure 3 shows that NaS inhibits glucose production from an aqueous extract of chow by the intestinal
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Fig. 3. Inhibition by sodium saccharin o f glucose production from an aqueous extract of laboratory chow by a small-intestinal fraction from rats. Each value is the mean o f three assays and range bars indicate the SEM.
fraction and therefore that the inhibition by NaS of hydrolysis of dietary carbohydrate is applicable to rat feeding studies., The demonstration that NaS can inhibit the enzymes responsible for dietary carbohydrate hydrolysis when coupled with its previously reported inhibition of the enzymes associated with hydrolysis of dietary protein (see Introduction) suggested a possil~le causality between the NaS dose-dependent enzyme inhibition and polydypsia. It was hypothesized that polydypsia was induced in order to provide sufficient intestinal fluid for lower bowel fermentation of the increased dietary components made available to the lower GI tract. This hypothesis was tested by ascertaining whether NaS ingestion altered the dry mass of the caecal contents and by determining whether systemic NaS (given iv) altered water consumption. Table 1 shows that while ingestion of NaS at 7.5% in the diet or 4% in drinking-water caused a doubling of water consumption and a greater than two-fold increase in the relative mass (g/kg body weight) of the caecal contents, it caused only a small increase in the water content of the caecal contents. In contrast, rats infused iv with 2.7 or 4.0 g NaS/kg body weight/day (equivalent to the systemic NaS load induced by 7.5 or 11.25% dietary NaS) for 5 days did not consume significantly more water than controls. Thus it can be concluded that ingestion of
NaS induces polydypsia in order to provide water for the hydration of the caecal contents; there is no such response to NaS given systematically. In order to ascertain whether the increased caecal fermentation that results from NaS ingestion alters the end-products of fermentation that are delivered to the host the lactate and VFA concentrations in caecal contents and their delivery to a medium during incubation in vitro were compared in caeca from control rats and rats given a diet containing 5% NaS. NaS increased the caecal concentration of lactate and decreased the caecal concentration of VFAs. The transport into the medium of these end-products of fermentation reflected the caecal concentrations (Fig. 4a, b). Comparison of the concentration of the individual VFAs in the medium and the caecal contents showed that NaS did not alter the acetate level but decreased the propionate (C3) and especially the butyrate (C4) and the valerate (C5) levels (Fig. 5). Filtrates (0.2-#m filter) of the caecal contents and media following incubation showed no mutagenic activity in Salmonella tester strains TA98 and TA100 with or without microsomal activation (E. D. Thompson, personal communication, 1982). There were no significant differences between male and female rats in any of the above observations of the effects of NaS on the GI tract. The above findings led to the hypothesis that the
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460
R. L. ANDERSON Table 1. Water consumption and caecal parameters in male rats given sodium saccharin in the diet or by iv infusion
Treatment Control: NaS:
7.5% dietary cellulose 7.5% in diet 4% in drinking-water 2.7 g/kg body weight/day, iv 4.0 g/kg body weight/day, iv 0.9% (w/v) aqueous solution, iv
NaCI:
Water consumption (g/g diet consumed)
Relative caecal weight g/kg body weight)
Caecal water (g/ 100 g of caecal contents)
pH of caecal contents
1.7 + 0.04 3.3 + 0.2 3.0 -I- 0.4 2.1 ___0.1 2.0 + 0.1 1.8 __.0.1
29 + 2 62 + 3 75 + 24 15 ___2 ND 18 + 1
78 83 84 ND ND ND
5.9 5.9 6.0 ND ND ND
NaS = Sodium saccharin ND = Not determined Each value is the mean of five or six determinations ( + S E M for water consumption and relative caecal weight). NaS was given in the diet or in drinking-water for 28 days, and iv infusion was carried out for 5 days. Infusion with 2.7 or 4.0 g NaS/kg body weight/day provided a systemic load of NaS equivalent to that induced by 7.5 or 11.5% NaS given in the diet.
polyuria and increased concentration of ions (CI-, PO34- , SO42-, Ca 2÷, Mg 2+ and Na +) in the urine that have been observed in rats fed NaS (Anderson, 1979; Schoenig & Anderson, 1985; Schoenig et aL 1985) result from increased caecal absorption of these ions and their passive transport into the urine and are not a specific response to renal clearance of NaS. To test this hypothesis the effect of iv-administered NaS and NaC1 on plasma concentrations and urinary excretion of selected minerals was ascertained. Table 2 shows that the administration iv of 11 mmol NaS/kg o
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body weight/day (the systemic load associated with a dietary level of 7.5% NaS) for 5 days results in plasma concentrations and urinary excretions (#mol/g diet ingested) of Ca, Mg, Na and P that are similar to those in rats given an equivalent iv dose of NaCL These values are not greatly different from those recorded in non-infused controls except for a higher urinary excretion of Na, undoubtedly due to the high dose being given. More than 95% of the infused saccharin was excreted in the urine. Thus, it can be concluded that the increased urinary excretion of ions that has been noted in rats ingesting NaS is due to increased absorption of these ions from the GI tract and is not a consequence of inhibition of their reabsorption during the renal clearance of NaS. Elsewhere in this issue it is reported that ingestion of NaS is associated with an increase in the relative bladder mass (g/kg body weight) that correlates with NaS-induced polyuria (Schoenig & Anderson, 1985). This work also shows that increased bladder mass was accompanied by an increase in the concentration (#moi/g dry tissue) of several cations in the bladder tissues of male rats but not of female rats (Schoenig & Anderson, 1985). The effect of 5 days of infusion of NaS or NaCI (c. 11 mmol/kg body weight/day) on bladder mass and cation concentrations in male rats
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Gastro-intestinal effects of saccharin in rats
461
Table 2. Mineral levels in plasma and urine o f male rats infused iv with sodium saccharin o r s o d i u m chloride Plasma* o r urinet levels o f Treatment
Ca
Mg
Masma 2.28 _+ 0.05 0.79 + 0.05 2.30 _+ 0.04 0.74 _+ 0.03
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4.2 + 0.2 4.5 __.0.3
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P
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4.26 _ 0.22 3.56 _ 0.23
302 + 28 296 +_ 32
37 _+ 4 36 _+ 6
*Values are for m e a n concentrations (raM) __. S E M for six samples taken after 5 days o f iv infusion. "tValues are for m e a n levels (/tmol/g diet consumed) _+ S E M for five daily determinations on six rats/treatment. Sodium saccharin and s o d i u m chloride were infused at 11 m m o l / k g body weight/day at a rate o f 14.4 ml/rat/day.
was measured to ascertain whether the responses noted in rats ingesting NaS were a specific response to high systemic levels of NaS or were a response to polyuria. There was no decrease in water consumption (Table 1) by rats infused with NaS or NaCI at a rate of 14.4ml/day. Therefore, this treatment does cause polyuria, approximately doubling urinary volume in comparison with non-infused controls. Figure 6 shows that dosing iv with either NaS or
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Sodium soechorin (g/kg/doy) Fig. 6. Effect on relative dry bladder mass (mg/kg body weight) of giving sodium saccharin (m) in the diet (7.5~) or by iv infusion (2.7 g/kg body weight/day) to male rats in comparison with that in control rats (ll) given basal diet or iv infusions of sodium chloride (0.9~ aqueous solution). Each rat weighed c. 250 g; sodium saccharin was given in the diet for 3 wk and iv infusion of sodium saccharin or sodium chloride was carried out for 5 days using an infusion volume of 14.4ml/rat/day. The systemic load of sodium saccharin was approximately equivalent for the orally and iv-dosed rats (c. 11 mol/kg body weight/day). Values are means for 5 rats and range bars indicate the SEM.
NaC1 for only 5 days results in a greater increase in relative dry bladder mass (mg/kg body weight) than does 3 wk of ingestion of a diet containing 7.5~ NaS (a concentration that results in a systemic NaS load of c. 11 mmol/kg body weight/day). The effects of these iv doses of NaS and NaCI on ion concentration in bladder tissue ~ m o l / g dry tissue) in comparison with the effect of 21 days of ingesting 7.5~ NaS are summarized in Fig. 7. Administration iv of either NaS or NaCI for only 5 days resulted in greater ion accumulation in bladder tissue than did 21 days of 7.5~ dietary NaS. This work allows three conclusions: (1) ion accumulation in the bladder is not dependent upon increased systemic loads of the ions involved (see Table 2); (2) mineral accumulation in the bladder is not dependent upon high urinary levels of NaS; (3) increased bladder mass in response to polyuria is a sufficient stimulus to induce ion accumulation in bladder tissue ( N a C 1 ) NaS). Since all of the bladder tumours that have been reported in rats ingesting NaS in chronic studies have occurred in the bladder epithelium (Arnold, Moodie, Grice et al. 1980; Schoenig et al. 1985; Taylor, Weinberger & Friedman, 1980; Tisdel, Nees, Harris & Derse, 1974), the site of the mineral accumulation in the bladders of male rats ingesting NaS at 7.5~o in the diet was ascertained by preparing an epithelial rich fraction (ERF) by scraping the luminal surface of everted bladders. The mineral concentrations in the ERF and total bladders from control rats and those ingesting 7.5~ NaS are shown in Fig. 8. The ERF fractions from the rats ingesting NaS was 9.5 _+0.4~o (mean _+ SEM) of the tissue total dry mass (ERF plus residue) compared with 7.9 _+ 1.3~o for the controls. In the control samples the concentration of each of the minerals determined except Na and Zn was higher in the ERF than in the whole bladder tissue. The mineral concentrations in the ERF from the rats fed NaS were higher than those in the controls. In fact while the ERF constituted only c. 10~ of the tissue dry mass, it contained the majority of the increased minerals associated with NaS ingestion. Discussion
The major purpose of the work described in this report was to ascertain whether the effects on the
462
R . L . ANDERSON
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Fig. 7. Mineral concentrations in the bladders of male rats given sodium saccharin in the diet (7.5~) for 3 wk ([]) or by iv infusion (2.7 g/kg body weight/day) for 5 days (~l) in comparison with those in control rats fed the basal diet (ll) or infused iv for 5 days with a 0.9~ aqueous solution of sodium chloride ([]). Each rat weighed c. 250 g. The systemic load of sodium saccharin was approximately equivalent for the orally and iv-dosed rats (c. 11 mmol/kg body weight/day). Each value is the mean for five rats (feeding study) or four rats (iv study) and range bars indicate the SEM. urine and bladder that have been observed in rats fed NaS in chronic studies could be shown to be a consequence of changes in dietary nutrient metabolism in the GI tract induced by the NaS. The results reported here are clearly consistent with the concept that the primary site of NaS effects in the rat is in the GI tract and that the alterations in the urine and bladder tissue are secondary responses. However, more important is the examination of the effects observed for their possible contribution to epithelial tumorigenesis in the rat bladder. An increase in the incidence of urinary bladder tumours has been observed in male rats fed NaS at ) 3 ~ in the diet and in two-generation studies the tumour incidence was greater in F, than in F0 rats (Arnold et No
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al. 1980; Schoenig et al. 1985; Taylor et al. 1980; Tisdel et al. 1974). In order for a condition to be
considered as a potential 'causative' factor in bladder tumorigenesis, that factor should show a response to NaS similar to that of the tumorigenesis. In this study no differences were observed between male and female rats in the GI effects of NaS, and neither has a greater response been observed in Ft than in F 0 rats (data not shown). Therefore the changes described in this report are not sufficient to be considered as the 'causal' factor(s) in bladder tumorigenesis. This does not mean, however, that the changes noted are not 'necessary' conditions for tumorigenesis. The fact that dietary NaS produces a male-specific mineral accumulation in the bladder fraction contain-
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froction
Fig. 8. Mineral concentrations in the 'epithelial-rich fraction' (ERF) of the bladders of control rats (m) and rats fed sodium saccharin at 7.5~o in the diet for 3 wk (m), in comparison with those in the whole bladder tissue (TB) from comparable rats. The ERF was prepared by scraping the bladder surface with a glass slide. The ERF values are means of three determinations on ERF pooled from the bladders of three rats and the TB values are the means of 5 samples. All samples were obtained from c. 7-wk-old male CR (CD) rats.
Gastro-intestinal effects of saccharin in rats ing the epithelium is the most pertinent observation reported, in that this response occurs in the same sex and at the same organ site in which tumorigenicity is noted. The fact that the male-specific mineral accumulation in the bladder is rapidly induced by the ingestion of high doses of NaS and that this response is no more pronounced in F1- than in F0-generation animals would certainly preclude the conclusion that mineral accumulation in the bladder is the 'cause' of bladder tumours in rats fed high doses of NaS in chronic studies. Furthermore, even if mineral accumulation in the bladder is necessary for tumorigenesis, the observation that iv-administered NaCI is as effective as iv-administered NaS in inducing mineral accumulation in the bladder would suggest that any c o m p o u n d that causes increased water absorption and polyuria should be able to induce bladder tumors in male rats. In this regard, it should be noted that analysis of the results o f a recent bioassay of NaS has revealed a strong correlation between polyuria early in life and the subsequent development of bladder turnouts a m o n g rats fed NaS at 7.5% in the diet (Schoenig et al. 1985). In summary, the studies reported show that NaSassociated changes in urine composition, increased bladder-tissue mass, and the male-specific mineral accumulation in bladder tissue in rats are consequences of the changes in GI metabolism that are associated with NaS-induced inhibition of the enzymes that digest dietary proteins and carbohydrates. While these findings have not identified a causal factor(s) for NaS-associated bladder tumorigenesis in rats, they do provide a mechanism for comparison between species of the effects of NaS at its primary site of activity.
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