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Urinary and bladder responses to immobilization in male rats
Fd Chem. Toxic. Vol. 28, No. 8, pp. 543-545, t990
0278-6915/90 $3.00 + 0.00 Copyright ~ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
U R I N A R Y A N D B L A D D E R RESPONSES TO IMMOBILIZATION IN MALE RATS R. L. ANDERSON, F. R. LEFEVER*, W. R. FRANCISand J. K. MAURER The Procter & Gamble Company, Miami Valley Laboratories, PO Box 398707, Cincinnati, OH 45239-8707, USA (Received 13 March 1990; revisions received 23 May 1990)
Abstract--lmmobilization of groups of five to nine male rats for 2-5 days results in a 50% increase in urinary bladder fresh weight compared with normally caged controls. The increase in urinary bladder weight was not due to tissue oedema and was accompanied by epithelial hyperplasia in some urinary bladders. Immobilization did not alter total urine volume, but it did decrease the frequency of urine voiding and doubled the mean urine weight/voiding. Thus, bladder distention caused by the increased volume per voiding caused a rapidly induced increase in bladder tissue growth, and was accompanied by an increase in bladder epithelial cell division.
INTRODUCTION
As part of a programme to ascertain the role of diuresis in urinary bladder carcinogenicity in rats ingesting high doses of sodium saccharin (NaS), it was noted that bladder weight was directly proportional to diuresis (Anderson, 1988). In the course of these latter studies, it was observed that rats immobilized for 5 days for iv infusions of NaS/saline showed marked increases in bladder weight when infused with saline (Anderson, 1985). In subsequent experiments, immobilization without infusion resulted in an increase in bladder weight (R. L. Anderson, unpublished observations, 1984). This result prompted an examination of the effect of immobilization (2-5 days) without fluid infusion on the urine and bladders of male rats. MATERIALSAND METHODS All studies used male Charles River CD rats derived from the Sprague-Dawley strain (Charles River Breeding Laboratories, Portage, MI, USA). Rats were purchased as young adults and they weighed 200-350 g at initiation of the studies. Rats were housed in a temperature- and humiditycontrolled environment (72 __+5°F; 50 + 20% relative humidity) with a 12-hr light/dark cycle. In all studies the rats were acclimatized to this environment for several days before assignment to experimental groups. During immobilization all rats had free access to feed pellets (Purina Laboratory Chow (no. 5001), Ralston Purina Co., St Louis, MO, USA) and distilled water. Feed and water consumption records were maintained during the immobilization period and body weights were recorded at the beginning and end of this time. *To whom all correspondence should be addressed. Abbreviations: NaS = sodium saccharin; NTA = nitrilotriacetic acid.
Immobilization units were of local design and consisted of a wire-mesh base with a curved wiremesh cover held in place by springs that restricted the movement of the rats. In addition, the rat's hind legs were tied to the cage bottom to further prevent movement. In some studies the urine voided in each 24-hr period was collected in flasks. When timed urine collections were made, the rats were housed in metabolism cages mounted over fraction collectors which collected urine in 64-min periods. Each fraction was weighed and only those fractions weighing >0.1 g were considered unique voidings and those <0.1 g were added to the tube preceding, or following, in calculating the number of voidings and the weight/voiding. In the timed collection study and when urine samples were analysed by atomic absorption, anal cups were used to prevent faecal contamination of the urine. The rats were killed by anaesthetization with CO2 and exsanguination; the urinary bladders were excised, rinsed with distilled water, blotted lightly and weighed. When the bladders were evaluated for epithelial-cell hyperplasia, they were fixed in buffered formalin, prepared for histological evaluation, and microscopically examined as previously described (Anderson et aL, 1988). In some studies the bladders were dried to a constant weight in a vacuum oven at ~40°C and the dry weight recorded. The dried bladders and pooled urine samples collected after 4 days and 5 days immobilization, respectively, were assayed for their content of Na, K, Ca, Mg, Zn and total P by atomic absorption spectroscopy as previously described (Anderson, 1979). RESULTS
In each study the rats that were immobilized lost body weight during the study and the controls gained body weight except during the 4-day immobilization when the controls also showed a slight weight loss (Tables 1-4). The loss of body weight varied because of the differences in immobilization time. Water 543
544
R.L. ANDERSONet al.
Table 1. Urinary and bladder responses of male rats to 2 days of immobilization Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine output (g/day) Bladder Weight (mg) Relative weight (mg/kg)t Hyperplasia (incidence)
Control +20 ± 2 23 +__2 35 _+ 2 17 + 2 84 + 2 348 +- 7 0/5
Immobilized - 13 ± 2 19 _+ 1 33 + 5 15 _+ 2 104 ± 8 475 ± 27 4/9
*Initial body weight, 225 +- 5 g. *Bladder weight/initial body weight. Values are means + SEM for five controls or nine immobilized rats.
consumption by the immobilized rats was lower than that of the controls in some studies but not in others. Urine output was generally comparable for the immobilized and control rats except for rats immobilized for 3 days with timed urine collections when immobilized rats had a greater urine output than control rats (Table 2). In every study immobilization was associated with an increase in the absolute weight of the fresh bladder tissue compared with the bladder weights of matched controls (Tables 1-4). Generally the fresh bladder tissue weight was increased by about 50% by immobilization and the increase was similar for most immobilization times except after immobilization for 2 days when the increase was only 24%. In those studies in which the dry weight of the bladder tissue was measured, immobilization did not alter the tissue moisture content (Tables 3 and 4). Thus immobilization was accompanied by an increase of about 50% in dry bladder weight as well as fresh tissue weight. On microscopic examination of bladders from five controls and nine rats immobilized for 2 days, four bladders from immobilized rats showed simple, diffuse epithelial-cell hyperplasia (Table 1). The bladders with epithelial-cell hyperplasia were heavier (120 _+ 12mg; mean_+SEM) than those without epithelial-cell hyperplasia from immobilized rats (96 _+ 5 rag) or controls (84 +_ 2 mg). When the urine output was collected in 64-min timed fractions for 3 days, it was observed that immobilization resulted in approximately half the number of voidings/day and that the weight of urine/voiding was roughly doubled (Table 2). The change in urination pattern was altered immediately by immobilization and it did not change again during the 3 days studied. Table 2. Urinary and bladder responses of male rats to 3 days of immobilization with timed urine collection* Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine Output Voidings/day Weight/voiding (g) Bladder Weight (mg) Relative weight (mg/kg)~
Control
Immobilized
+ 15 + 2 23 + 1 35 ± 2
- 2 9 _+_4 19 + 1 40 + 8
15.8 z 0.8 17 + 0.4 0.9 _+ 0.04
15.9 + 1.3 10 +- 0.8 2.2 + 0.2
82 t 7 282 + 9
120 + 12 413 +- 31
*Urine was collected in 64-min fractions. *Initial body weight 269 ± 19 g. SBladder weight/initial body weight. Values are means ± SEM for groups of eight rats.
Table 3. Effect of 4 days of immobilization on clinical parameters and bladder ion concentrations in male rats Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine output (g/day) Bladder weight Fresh (mg) Dry (mg) Relative weight (mg/kg)'t" Bladder ions (/zmol/g dry tissue) Na K Ca Mg Zn P
Control
Immobilized
- 5 +- 1 16.7 ± 0.2 32 _+ 3 18 +- I
36 +- 2 17.0 +- 0.2 26 +- I 17 + 1
96 + 7 18 + I 414 +- 30
146 +- 21 27 + 3 618 + 73
260 z 6 397 t 15 9.2±0.3 31.0 + 0.3 1.7 ± 0.1 238_+3
289 ± 6 405 + 13 12.8+-1.8 33.7+0.4 1.9+-0.1 261 +-3
*Initial body weight 232 + 2 g. *Bladder weight (fresh)/initial body weight. Values are means_+SEM for four controls or five immobilized rats.
Because we had noted that feeding NaS at doses that are carcinogenic to the bladder was associated with increases in both bladder-tissue and urine ion concentrations (Schoenig and Anderson, 1985), the effect of immobilization on bladder-tissue and urinary ions was determined (Tables 3 and 4). Immobilization for 4 days was associated with small increases in bladder-tissue ion levels with the exception of a large increase in tissue Ca (Table 3). The results for urinary ions (Table 4) established that immobilization for 5 days was associated with a slight increase in urinary excretion of Na, an increase in K excretion and a decrease in Mg excretion per g of diet ingested. Immobilization decreased urinary Ca output to approximately a quarter of the control value and increased urinary P excretion per g diet ingested four-fold compared with the control value. Immobilization did not alter urinary pH or osmolality (Table 4). DISCUSSION
The results demonstrate that the rat urinary bladder can undergo an almost 50% increase in weight in as little as 3 days while the rat is losing body weight. The increase in bladder weight is not due to Table 4. Effect of 5 days immobilization on clinical parameters and urine characteristics in male rats Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine Weight (g/day) pH Osmolality (mosmol/litre) Urine ions (l~mol/g diet) Na K Ca Mg P Bladder Fresh weight (mg) Dry weight (rag) Relative weight (mg/kg)t
Control
Immobilized
+31 ± 2 23 + 0.4 35 ±. 1
18 + 3 17 + 0.6 30 + 3
12.6+0.5 7.9 + 0.1 2022 ± 52
13.0+ 1.8 8.0_+0.1 2018 ± 75
107+4 75_+4 3.8 ~ 0.6 14+2 9L2
117+_6 103+7 1.0 + 0.2 10+-3 38_+7
86 ± 4 17 ± 1 391 ± 15
130 + 21 27 +- 3 597 _+ 96
*Initial body weight 219 + 2 g. +Bladder weight (fresh)/initial body weight. VMues are means±SEM for groups of five rats.
Immobilization and rat urine/bladder oedema and it is accompanied by diffuse, simple hyperplasia of the epithelium in some rats. Because the epithelium constitutes only a small portion of the total bladder weight and because immobilization did not greatly increase the number of epithelial cell layers (only four of nine samples showed increased numbers of epithelial cells), most of the weight increase must reflect growth of the smooth muscle of the tunica muscularis. The results of the timed urine collection study indicated that the rapid increase in bladder weight probably resulted from the increased urine weight per voiding that accompanied the immobilization. This association between increased bladder weight and the increased weight per voiding is consistent with earlier results, which showed that there was a linear correlation between bladder weight and urine output over the range of 20g/kg body weight to 120g/kg body weight (Anderson, 1988). With the immobilization model, the rapidly induced increase in bladder weight is not associated with an increase in total urine output but correlates with the output/voiding. The fact that the response was almost as great after 48 hr as after 120 hr of immobilization (Tables 1 and 4) shows that the bladder response was very rapid. This finding is consistent with a report which demonstrated that bladder distension by saline instillation caused a wave of DNA synthesis in the bladder (Martin, 1962). The histological evaluation of bladders after 48 hr of immobilization established that the rapid bladderweight increase was associated with some epithelial hyperplasia, although this was a mild and diffuse response and was observed in only four of the nine bladders examined. The findings of this study may have some implications for the interpretation of reports on the effects of sodium saccharin on changes of bladder morphology in short-term studies. Based on scanning electron microscopy studies, it has been suggested that bladders from rats ingesting NaS have areas of surface cell loss and increased numbers of immature cells on the bladder surface (Fukushima and Cohen, 1980; Murasaki and Cohen, 1981). In contrast to the cell-loss hypothesis, it can be speculated that the increase in urine volume that accompanies NaS ingestion causes separations in the surface epithelium in the bladder due to over-distention of the organ followed by an increased cell division to repair the lesions. No data are available that indicate that diuresis caused by NaS is accompanied by increased epithelial-cell turnover in the bladder. Increased epithelial cell division does occur and, thus, the recovery model seems as plausible as the surface cell loss model for the presence of immature cells on the bladder surface. In contrast, the concept of surface cell loss followed by repair can be found in studies with nitrilotriacetic acid (NTA; Anderson et al., 1985) and tissue explants grown in a low-Ca medium (Reese and
545
Friedman, 1978). It has been demonstrated that NTA causes surface cell loss when the urinary NTA concentration exceeds that of the urinary divalent cations due to removal of the extracellular Ca faster than it can be replaced by diffusion from the circulation. N T A causes bladder growths that are endophytic (Anderson and Alden, 1989) by contrast with NaS, which produces exophytic or papillomatous growths (Schoenig et al., 1985). This implies that surface cell loss results in cellular proliferation resulting in endophytic growth rather than papillomatous growth as noted with diuresis. The hypothesis that the type of surface damage dictates the growth orientation of subsequent cellular proliferative lesions requires additional study and the immobilization model provides a means of examining this concept. REFERENCES
Anderson R. L. (1979) Response of male rats to sodium saccharin ingestion: urine composition and mineral balance. Fd Cosmet. Toxicol. 17, 195-200. Anderson R. L. (1985) Some changes in gastro-intestinal metabolism and in urine and bladders of rats in response to sodium saccharin ingestion. Fd Chem. Toxic. 23, 457-463. Anderson R. L. (1988) An hypothesis of the mechanism of urinary bladder tumorigenesis in rats ingesting sodium saccharin. Fd Chem. Toxic. 26, 637-644. Anderson R. L. and Alden C. L. (1989) Risk assessment for nitrilotriacetic acid (NTA). In The Risk Assessment o f Environmental Hazards. A Textbook o f Case Studies.
Edited by D. J. Pauftenbach. pp. 390-426. John Wiley, New York. Anderson R. L., Bishop W. E. and Campbell R. L. (1985) A review of the environmental and mammalian toxicology of nitrilotriacetic acid. C R C Crit. Rev. Toxicol. 15, 1-102. Anderson R. L., Lefever F. R. and Maurer J. K. (1988) The effect of various saccharin forms on gastro-intestinal tract, urine and bladder of male rats. Fd Chem. Toxic. 26, 665-669. Fukushima S. and Cohen S. M. (1980) Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res. 40, 734-736. Martin B. F. (1962) The effect of distension of the urinary bladder on the lining epithelium and on its histochemical reaction for alkaline phosphatase. Ann. Histochim. 7, 51-61. Murasaki G. and Cohen S. M. (1981) Effects of dose sodium saccharin on the induction of rat urinary proliferation. Cancer Res. 41, 942-944. Reese D. H. and Friedman R. D. (1978) Suppression of dysplasia and hyperplasia by calcium in organ-cultured urinary bladder epithelium. Cancer Res. 38, 586-592. Schoenig G. P. and 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, 465-474. Schoenig G. P., Goldenthal E. I., Geil R. G., Frith C. H., Richter W. R. and Carlborg F. W. (1985) Evaluation of the dose response and in utero exposure to saccharin in the rat. Fd Chem. Toxic. 23, 475-490.
0278-6915/90 $3.00 + 0.00 Copyright ~ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
U R I N A R Y A N D B L A D D E R RESPONSES TO IMMOBILIZATION IN MALE RATS R. L. ANDERSON, F. R. LEFEVER*, W. R. FRANCISand J. K. MAURER The Procter & Gamble Company, Miami Valley Laboratories, PO Box 398707, Cincinnati, OH 45239-8707, USA (Received 13 March 1990; revisions received 23 May 1990)
Abstract--lmmobilization of groups of five to nine male rats for 2-5 days results in a 50% increase in urinary bladder fresh weight compared with normally caged controls. The increase in urinary bladder weight was not due to tissue oedema and was accompanied by epithelial hyperplasia in some urinary bladders. Immobilization did not alter total urine volume, but it did decrease the frequency of urine voiding and doubled the mean urine weight/voiding. Thus, bladder distention caused by the increased volume per voiding caused a rapidly induced increase in bladder tissue growth, and was accompanied by an increase in bladder epithelial cell division.
INTRODUCTION
As part of a programme to ascertain the role of diuresis in urinary bladder carcinogenicity in rats ingesting high doses of sodium saccharin (NaS), it was noted that bladder weight was directly proportional to diuresis (Anderson, 1988). In the course of these latter studies, it was observed that rats immobilized for 5 days for iv infusions of NaS/saline showed marked increases in bladder weight when infused with saline (Anderson, 1985). In subsequent experiments, immobilization without infusion resulted in an increase in bladder weight (R. L. Anderson, unpublished observations, 1984). This result prompted an examination of the effect of immobilization (2-5 days) without fluid infusion on the urine and bladders of male rats. MATERIALSAND METHODS All studies used male Charles River CD rats derived from the Sprague-Dawley strain (Charles River Breeding Laboratories, Portage, MI, USA). Rats were purchased as young adults and they weighed 200-350 g at initiation of the studies. Rats were housed in a temperature- and humiditycontrolled environment (72 __+5°F; 50 + 20% relative humidity) with a 12-hr light/dark cycle. In all studies the rats were acclimatized to this environment for several days before assignment to experimental groups. During immobilization all rats had free access to feed pellets (Purina Laboratory Chow (no. 5001), Ralston Purina Co., St Louis, MO, USA) and distilled water. Feed and water consumption records were maintained during the immobilization period and body weights were recorded at the beginning and end of this time. *To whom all correspondence should be addressed. Abbreviations: NaS = sodium saccharin; NTA = nitrilotriacetic acid.
Immobilization units were of local design and consisted of a wire-mesh base with a curved wiremesh cover held in place by springs that restricted the movement of the rats. In addition, the rat's hind legs were tied to the cage bottom to further prevent movement. In some studies the urine voided in each 24-hr period was collected in flasks. When timed urine collections were made, the rats were housed in metabolism cages mounted over fraction collectors which collected urine in 64-min periods. Each fraction was weighed and only those fractions weighing >0.1 g were considered unique voidings and those <0.1 g were added to the tube preceding, or following, in calculating the number of voidings and the weight/voiding. In the timed collection study and when urine samples were analysed by atomic absorption, anal cups were used to prevent faecal contamination of the urine. The rats were killed by anaesthetization with CO2 and exsanguination; the urinary bladders were excised, rinsed with distilled water, blotted lightly and weighed. When the bladders were evaluated for epithelial-cell hyperplasia, they were fixed in buffered formalin, prepared for histological evaluation, and microscopically examined as previously described (Anderson et aL, 1988). In some studies the bladders were dried to a constant weight in a vacuum oven at ~40°C and the dry weight recorded. The dried bladders and pooled urine samples collected after 4 days and 5 days immobilization, respectively, were assayed for their content of Na, K, Ca, Mg, Zn and total P by atomic absorption spectroscopy as previously described (Anderson, 1979). RESULTS
In each study the rats that were immobilized lost body weight during the study and the controls gained body weight except during the 4-day immobilization when the controls also showed a slight weight loss (Tables 1-4). The loss of body weight varied because of the differences in immobilization time. Water 543
544
R.L. ANDERSONet al.
Table 1. Urinary and bladder responses of male rats to 2 days of immobilization Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine output (g/day) Bladder Weight (mg) Relative weight (mg/kg)t Hyperplasia (incidence)
Control +20 ± 2 23 +__2 35 _+ 2 17 + 2 84 + 2 348 +- 7 0/5
Immobilized - 13 ± 2 19 _+ 1 33 + 5 15 _+ 2 104 ± 8 475 ± 27 4/9
*Initial body weight, 225 +- 5 g. *Bladder weight/initial body weight. Values are means + SEM for five controls or nine immobilized rats.
consumption by the immobilized rats was lower than that of the controls in some studies but not in others. Urine output was generally comparable for the immobilized and control rats except for rats immobilized for 3 days with timed urine collections when immobilized rats had a greater urine output than control rats (Table 2). In every study immobilization was associated with an increase in the absolute weight of the fresh bladder tissue compared with the bladder weights of matched controls (Tables 1-4). Generally the fresh bladder tissue weight was increased by about 50% by immobilization and the increase was similar for most immobilization times except after immobilization for 2 days when the increase was only 24%. In those studies in which the dry weight of the bladder tissue was measured, immobilization did not alter the tissue moisture content (Tables 3 and 4). Thus immobilization was accompanied by an increase of about 50% in dry bladder weight as well as fresh tissue weight. On microscopic examination of bladders from five controls and nine rats immobilized for 2 days, four bladders from immobilized rats showed simple, diffuse epithelial-cell hyperplasia (Table 1). The bladders with epithelial-cell hyperplasia were heavier (120 _+ 12mg; mean_+SEM) than those without epithelial-cell hyperplasia from immobilized rats (96 _+ 5 rag) or controls (84 +_ 2 mg). When the urine output was collected in 64-min timed fractions for 3 days, it was observed that immobilization resulted in approximately half the number of voidings/day and that the weight of urine/voiding was roughly doubled (Table 2). The change in urination pattern was altered immediately by immobilization and it did not change again during the 3 days studied. Table 2. Urinary and bladder responses of male rats to 3 days of immobilization with timed urine collection* Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine Output Voidings/day Weight/voiding (g) Bladder Weight (mg) Relative weight (mg/kg)~
Control
Immobilized
+ 15 + 2 23 + 1 35 ± 2
- 2 9 _+_4 19 + 1 40 + 8
15.8 z 0.8 17 + 0.4 0.9 _+ 0.04
15.9 + 1.3 10 +- 0.8 2.2 + 0.2
82 t 7 282 + 9
120 + 12 413 +- 31
*Urine was collected in 64-min fractions. *Initial body weight 269 ± 19 g. SBladder weight/initial body weight. Values are means ± SEM for groups of eight rats.
Table 3. Effect of 4 days of immobilization on clinical parameters and bladder ion concentrations in male rats Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine output (g/day) Bladder weight Fresh (mg) Dry (mg) Relative weight (mg/kg)'t" Bladder ions (/zmol/g dry tissue) Na K Ca Mg Zn P
Control
Immobilized
- 5 +- 1 16.7 ± 0.2 32 _+ 3 18 +- I
36 +- 2 17.0 +- 0.2 26 +- I 17 + 1
96 + 7 18 + I 414 +- 30
146 +- 21 27 + 3 618 + 73
260 z 6 397 t 15 9.2±0.3 31.0 + 0.3 1.7 ± 0.1 238_+3
289 ± 6 405 + 13 12.8+-1.8 33.7+0.4 1.9+-0.1 261 +-3
*Initial body weight 232 + 2 g. *Bladder weight (fresh)/initial body weight. Values are means_+SEM for four controls or five immobilized rats.
Because we had noted that feeding NaS at doses that are carcinogenic to the bladder was associated with increases in both bladder-tissue and urine ion concentrations (Schoenig and Anderson, 1985), the effect of immobilization on bladder-tissue and urinary ions was determined (Tables 3 and 4). Immobilization for 4 days was associated with small increases in bladder-tissue ion levels with the exception of a large increase in tissue Ca (Table 3). The results for urinary ions (Table 4) established that immobilization for 5 days was associated with a slight increase in urinary excretion of Na, an increase in K excretion and a decrease in Mg excretion per g of diet ingested. Immobilization decreased urinary Ca output to approximately a quarter of the control value and increased urinary P excretion per g diet ingested four-fold compared with the control value. Immobilization did not alter urinary pH or osmolality (Table 4). DISCUSSION
The results demonstrate that the rat urinary bladder can undergo an almost 50% increase in weight in as little as 3 days while the rat is losing body weight. The increase in bladder weight is not due to Table 4. Effect of 5 days immobilization on clinical parameters and urine characteristics in male rats Parameter Body-weight change (g)* Diet consumed (g/day) Water consumed (g/day) Urine Weight (g/day) pH Osmolality (mosmol/litre) Urine ions (l~mol/g diet) Na K Ca Mg P Bladder Fresh weight (mg) Dry weight (rag) Relative weight (mg/kg)t
Control
Immobilized
+31 ± 2 23 + 0.4 35 ±. 1
18 + 3 17 + 0.6 30 + 3
12.6+0.5 7.9 + 0.1 2022 ± 52
13.0+ 1.8 8.0_+0.1 2018 ± 75
107+4 75_+4 3.8 ~ 0.6 14+2 9L2
117+_6 103+7 1.0 + 0.2 10+-3 38_+7
86 ± 4 17 ± 1 391 ± 15
130 + 21 27 +- 3 597 _+ 96
*Initial body weight 219 + 2 g. +Bladder weight (fresh)/initial body weight. VMues are means±SEM for groups of five rats.
Immobilization and rat urine/bladder oedema and it is accompanied by diffuse, simple hyperplasia of the epithelium in some rats. Because the epithelium constitutes only a small portion of the total bladder weight and because immobilization did not greatly increase the number of epithelial cell layers (only four of nine samples showed increased numbers of epithelial cells), most of the weight increase must reflect growth of the smooth muscle of the tunica muscularis. The results of the timed urine collection study indicated that the rapid increase in bladder weight probably resulted from the increased urine weight per voiding that accompanied the immobilization. This association between increased bladder weight and the increased weight per voiding is consistent with earlier results, which showed that there was a linear correlation between bladder weight and urine output over the range of 20g/kg body weight to 120g/kg body weight (Anderson, 1988). With the immobilization model, the rapidly induced increase in bladder weight is not associated with an increase in total urine output but correlates with the output/voiding. The fact that the response was almost as great after 48 hr as after 120 hr of immobilization (Tables 1 and 4) shows that the bladder response was very rapid. This finding is consistent with a report which demonstrated that bladder distension by saline instillation caused a wave of DNA synthesis in the bladder (Martin, 1962). The histological evaluation of bladders after 48 hr of immobilization established that the rapid bladderweight increase was associated with some epithelial hyperplasia, although this was a mild and diffuse response and was observed in only four of the nine bladders examined. The findings of this study may have some implications for the interpretation of reports on the effects of sodium saccharin on changes of bladder morphology in short-term studies. Based on scanning electron microscopy studies, it has been suggested that bladders from rats ingesting NaS have areas of surface cell loss and increased numbers of immature cells on the bladder surface (Fukushima and Cohen, 1980; Murasaki and Cohen, 1981). In contrast to the cell-loss hypothesis, it can be speculated that the increase in urine volume that accompanies NaS ingestion causes separations in the surface epithelium in the bladder due to over-distention of the organ followed by an increased cell division to repair the lesions. No data are available that indicate that diuresis caused by NaS is accompanied by increased epithelial-cell turnover in the bladder. Increased epithelial cell division does occur and, thus, the recovery model seems as plausible as the surface cell loss model for the presence of immature cells on the bladder surface. In contrast, the concept of surface cell loss followed by repair can be found in studies with nitrilotriacetic acid (NTA; Anderson et al., 1985) and tissue explants grown in a low-Ca medium (Reese and
545
Friedman, 1978). It has been demonstrated that NTA causes surface cell loss when the urinary NTA concentration exceeds that of the urinary divalent cations due to removal of the extracellular Ca faster than it can be replaced by diffusion from the circulation. N T A causes bladder growths that are endophytic (Anderson and Alden, 1989) by contrast with NaS, which produces exophytic or papillomatous growths (Schoenig et al., 1985). This implies that surface cell loss results in cellular proliferation resulting in endophytic growth rather than papillomatous growth as noted with diuresis. The hypothesis that the type of surface damage dictates the growth orientation of subsequent cellular proliferative lesions requires additional study and the immobilization model provides a means of examining this concept. REFERENCES
Anderson R. L. (1979) Response of male rats to sodium saccharin ingestion: urine composition and mineral balance. Fd Cosmet. Toxicol. 17, 195-200. Anderson R. L. (1985) Some changes in gastro-intestinal metabolism and in urine and bladders of rats in response to sodium saccharin ingestion. Fd Chem. Toxic. 23, 457-463. Anderson R. L. (1988) An hypothesis of the mechanism of urinary bladder tumorigenesis in rats ingesting sodium saccharin. Fd Chem. Toxic. 26, 637-644. Anderson R. L. and Alden C. L. (1989) Risk assessment for nitrilotriacetic acid (NTA). In The Risk Assessment o f Environmental Hazards. A Textbook o f Case Studies.
Edited by D. J. Pauftenbach. pp. 390-426. John Wiley, New York. Anderson R. L., Bishop W. E. and Campbell R. L. (1985) A review of the environmental and mammalian toxicology of nitrilotriacetic acid. C R C Crit. Rev. Toxicol. 15, 1-102. Anderson R. L., Lefever F. R. and Maurer J. K. (1988) The effect of various saccharin forms on gastro-intestinal tract, urine and bladder of male rats. Fd Chem. Toxic. 26, 665-669. Fukushima S. and Cohen S. M. (1980) Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res. 40, 734-736. Martin B. F. (1962) The effect of distension of the urinary bladder on the lining epithelium and on its histochemical reaction for alkaline phosphatase. Ann. Histochim. 7, 51-61. Murasaki G. and Cohen S. M. (1981) Effects of dose sodium saccharin on the induction of rat urinary proliferation. Cancer Res. 41, 942-944. Reese D. H. and Friedman R. D. (1978) Suppression of dysplasia and hyperplasia by calcium in organ-cultured urinary bladder epithelium. Cancer Res. 38, 586-592. Schoenig G. P. and 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, 465-474. Schoenig G. P., Goldenthal E. I., Geil R. G., Frith C. H., Richter W. R. and Carlborg F. W. (1985) Evaluation of the dose response and in utero exposure to saccharin in the rat. Fd Chem. Toxic. 23, 475-490.