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

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

Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. I. Effects at 30 days post-birth

The incidence of sodium saccharin (NaS)-associated bladder tumours in male rats increases when exposure to high doses begins in utero or at birth compared with treatment after weaning. The present experiment evaluated the effect of NaS exposure on selected physiological parameters in young second generation rats. 6-wk-old male and female Sprague-Dawley rats were placed on either a diet supplemented with 7.5% NaS or an untreated diet, and mated 4-6 wk later. Treatment was continued through lactation and the offspring were weaned on to the same diet. Body weights were significantly depressed in NaS-treated litters by 4 days after birth, and were 35% lower than controls by 30 days when the animals were killed. NaS treatment of the offspring was associated with an increase in faecal moisture content and caecal content weight, changes in several urinary analytes, a 50% increase in serum cholesterol a 10-fold increase in serum triglycerides and decreases in serum and hepatic vitamins. In addition, NaS-treated dams and pups were anaemic. Relatively few differences between males and females were noted, but significant inter-litter differences existed. The numerous physiological changes indicate that 7.5% dietary NaS exceeds the maximum tolerated dose for weanling rats.     

Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet

The effects of the salt form of saccharin and of diet on urinary ion levels have been studied in rats. Sodium saccharin (NaS) or calcium saccharin (CaS) was fed at a level of 5% in either Agway Prolab 3200 diet or AIN-76 diet to male, 5-wk-old F344 rats for 10 wk. The AIN-76 diet contained considerably less calcium, sodium and potassium than the Prolab 3200 diet, and smaller amounts of these ions were eliminated over 24 hr in the urine of rats fed the AIN-76 diet. Although food consumption was less in the groups fed AIN-76, total urinary saccharinate ion excretion with either saccharin salt was comparable with, or even higher than, that excreted by rats fed either salt in the Prolab 3200 diet. Rats fed Prolab 3200 eliminated approximately equal amounts of saccharinate ion in the faeces and urine. Rats fed AIN-76 eliminated about 10-20 times as much saccharin in the urine as in the faeces. Total saccharin excretion (faecal and urinary) was not influenced by the salt form. Water intake and urine volume were lower in rats fed control AIN-76 diet in comparison with those fed Prolab 3200, and were increased above the control level in groups fed saccharin in the AIN-76 diet. Urine electrolyte levels and osmolality were lower in the groups fed AIN-76. In general, NaS administration in either diet resulted in increased urinary sodium compared with controls, and the pH was at, or above, the level of control rats. CaS resulted in increased urinary calcium and decreased pH. There were marked diurnal variations in the urinary excretion of the various electrolytes, pH, and urine volume over a 24-hr period in all rats. This diurnal variation was more pronounced in the rats fed the Prolab 3200 diet. These results indicate that NaS and CaS have marked effects on the excretion of urinary electrolytes, and that these effects are influenced by diet.     

Alterations in the rat kidney associated with sodium saccharin feeding

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

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

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

Effects of a saccharin and cyclamate mixture on rat embryos

Sodium saccharin (NaS) and calcium cyclamate (CaC) are artificial sweeteners widely used in food and drink. To evaluate their toxicological effects on preimplantation mammalian embryos, pregnant rats were gavaged with 1.65 mg NaS/kg bw + 3.85 mg CaC/kg bw (DI) or 6.6 mg NaS/kg bw + 15.4 mg CaC/kg bw (D2) on days 1, 2, 3 and 4 of pregnancy (positive vaginal smear = day 1). The female rats were killed on day 5 of the pregnancy (GD 5), maternal organs weighed, and the blastocysts collected, counted and evaluated for gross morphology, cell number and mitotic index. There was no alteration in maternal organ weights, but there was an increase of the cell number/embryo in the dams treated with that NaS + CaC mixtures (D1 = 37.20 +/- 7.96; D2 = 37.26 +/- 10.90) compared to control group (32.24 +/- 6.73). Embryos whose dams were exposed to NaS + CaC may have adapted for implantation into the uterus but more studies are needed to demonstrate this mechanism of action.     

Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. II. Dose response and reversibility

A previous study in our laboratory demonstrated that 30-day-old Sprague-Dawley rats exposed to 7.5% sodium saccharin (NaS) since conception differ from untreated rats in several physiological parameters. In the present study, to determine the dose response of the changes associated with NaS treatment, animals were evaluated at 30 days post-birth, after treatment with dietary levels of 0, 1, 3 or 7.5% NaS since conception. Most physiological consequences of NaS treatment in the weanling rat, including anaemia and reductions in serum folate and vitamin A concentrations, were dose dependent. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at the two lower doses of NaS but were significantly increased with 7.5% NaS. The no-effect level (NOEL) was similar for physiological effects and for bladder tumour production in two-generation studies (1% NaS in the diet). The reversibility of the effects of 7.5% NaS was examined in 90-day-old rats. The increases in lipids and vitamin E were reversible. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% dietary NaS on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumours.     

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

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

An hypothesis of the mechanism of urinary bladder tumorigenesis in rat ingesting sodium saccharin

An hypothesis is presented of a mechanism for the sodium saccharin (NaS)-associated tumorigenesis of the urinary bladder that occurs in male rats. The ingestion of high doses of NaS is associated with increased urine volume and bladder mass. In rats with an inherently high urine output, the diuresis associated with NaS ingestion combined with the increasing diuresis that occurs with age in male rats results in a chronic demand for a bladder-volume increase that is met by excessive cell division of the bladder epithelium. This enhanced mitosis in the bladder epithelium can result in a significant incidence of bladder tumours. Male rats exposed to NaS during early life show an exacerbation of tumour incidence, and it is proposed that this is because the exacerbation of the effects of NaS on the gastro-intestinal and urinary tracts results in increased urine output and bladder hyperplasia in these rats.     

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

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

Effects of opiate agonists and antagonists on fluid intake and saccharin choice in the rat

Both naloxone (3 and 10 mg X kg-1) and naltrexone (1-10 mg X kg-1) abolished the preference for a highly palatable 0.05% sodium saccharin solution in rats that had been adapted to a 22 hr water-deprivation schedule. The effect occurred as a result of a selective decrease in the consumption of the saccharin solution, since the intake of water, which was concurrently available in the two-fluid choice test, remained unaffected. When a less preferred saccharin solution was used (0.01%), naltrexone exerted a similar suppressant effect on the sodium preference, whilst naloxone failed to produce significant effects on the intake of saccharin solution or water. The data for the opiate agonists were interpreted in terms of a drug-induced blockade of the natural reward of highly palatable fluids in thirsty rats. In the same choice test, morphine and a stabilised enkephalin analogue, with a selective agonist action at mu-opiate receptors (RX783030), failed to influence the preference for the palatable saccharin solutions. In water-deprived animals, at least, exogenous opiate agonists, active at mu-receptors, did not appear to influence the reward of the palatable solutions.     

低钠与限钠对难治性心力衰竭的影响

目的 探讨心力衰竭患者低钠及限钠与难治性心力衰竭的关系。方法 将186例心力衰竭患者根据治疗情况分为一般心力衰竭和难治性心力衰竭两组，在入选时及1周后检查外周静脉血钠浓度。结果 一般心力衰竭组低钠的发生率为35．91％，难治性心力衰竭组低钠发生率为70．45％，两组发生率有显著性差异（P〈0．005）。限钠组难治性心力衰竭发生率为30．88％，非限钠组难治性心力衰竭发生率为19．49％，两组发生率有显著性差异（P〈0．05）。结论 过分限钠是引起低钠血症的重要原因，低钠是难治性心力衰竭存在的一大原因。     

Induction of DNA synthesis by sodium phenobarbital, uracil, and sodium saccharin in urinary bladder of the F344 rat

DNA synthesis in urothelial cells was determined 2, 4, 6, 10, and 16 weeks following dietary administration of promoters of bladder tumors to weanling male F344 rats. The experimental groups were fed AIN-76A diet or this diet containing 0.15% sodium phenobarbital, 3% uracil, 5% sodium saccharin, 2% sodium bicarbonate, or a combination of 5% sodium saccharin and 2% sodium bicarbonate. Two other groups were given Wayne rodent chow alone or in combination with 5% sodium saccharin. A group of rats given a drinking water containing 0.01% N-butyl-N-(4-hydroxybutyl)nitrosamine served as a positive control. With the exception of phenobarbital which increased the labeling index but did not induce hyperplasia, all other compounds increased the labeling index and induced hyperplasia. The combination of sodium saccharin and sodium bicarbonate was more effective than either compound alone in increasing the labeling index. Sodium saccharin was equally active when incorporated in either the AIN diet or the Wayne rodent chow. The present results suggest that a fundamental mechanism of bladder tumor promotion may be due to an increased DNA synthesis which leads to an increased turnover rate of urothelial cells rather than hyperplasia. However, since sodium saccharin is a promoter when incorporated into the Wayne diet but not into the AIN diet, the ability to stimulate DNA synthesis may be an important but insufficient condition for promotion by saccharin.     

The effect of four different types of diet on the bioavailability of loracarbef.

This randomized open-label single-dose crossover pharmacokinetic study was carried out to assess the effect of different diets on the bioavailability of loracarbef in 24 healthy male volunteers. A single dose of loracarbef in 200-mg tablet form was administered at 5 different times: after overnight fasting, after two vegetarian (high-fat and low-fat) diets, and following two non-vegetarian (high-fat and low-fat) diets. Serial blood samples were collected up to 10 h post-dose. Serum loracarbef concentrations were determined by a validated high performance liquid chromatographic (HPLC) method. Area under curve (AUC) values were significantly affected only by non-vegetarian diets; however the time to reach maximum serum concentration (Tmax) was prolonged and the maximum serum concentration (Cmax was decreased by all types of meals. The non-vegetarian diets affected the rate of absorption of loracarbef more than the vegetarian diets. The lowest decrease in Cmax was produced by the high-fat vegetarian diet, while the maximum was produced by the low-fat non-vegetarian diet. The results of this study indicate that while the rate of loracarbef absorption is significantly decreased by all diets, the extent of absorption is only reduced significantly by the non-vegetarian diets. The rate of elimination (k(el)) was not found to be significantly decreased by any of the diets. As compared to the high-fat non-vegetarian diet, the time beyond minimum inhibitory concentration (MIC90) concentration was significantly increased by the high-fat vegetarian diet. The implications of these findings for the large Indian vegetarian population are considerable.     

Effect of short-term administration of sodium saccharin on rhesus monkeys

Sodium saccharin (NaS) was incorporated into biscuits or the drinking-water and fed to rhesus monkeys at progressively increasing doses in order to determine the maximum dose that the monkeys would voluntarily consume and/or tolerate. Very little rejection of NaS-treated biscuits or drinking-water was observed. However, severe diarrhoea which precluded further treatment occurred when the dose of NaS reached 8.0% (approximately 1600 mg/kg body weight/day) in the biscuits and 0.48% (approximately 900–2400 mg/kg/day) in the drinking-water. An increase in fluid intake occurred in monkeys in both treatment groups. An increase in urine volume and a decrease in urine osmolality occurred in monkeys fed NaS in the drinking-water. No effects on body weight, food consumption or urine pH were observed in monkeys in either treatment groups. All animals rapidly recovered when they were given untreated biscuits or water. Therefore, although monkeys will voluntarily consume rather high doses of NaS incorporated into biscuits or drinking-water, adverse effects on their general well-being preclude such administration for more than a few days.     

Effects of Na saccharin feeding and urine on barrier properties of excised rat urinary bladder

When male rats of certain strains are fed a diet with 3% or more Na saccharin, their urinary bladders develop epithelial hyperplasia and a greater incidence of tumors. Since the daily dose of saccharin is high, a link between tumor formation and the disruption of urothelial physiologic and biochemical processes has been sought. We fed male and female Sprague-Dawley rats a saccharin-free or 7.5% Na saccharin diet for 1 month. Excised bladders were mounted in flux chambers and exposed to Krebs-Ringer bicarbonate solution (KRB) or urine. Bioelectric properties and 22Na, 36Cl, and [14C]mannitol or [3H]mannitol unidirectional fluxes were measured by conventional techniques. No differences were noted between bladders from male and female animals or between Na saccharin-fed animals and animals fed the saccharin-free diet. When both surfaces of the epithelium were exposed to KRB, transepithelial dc conductance fell over 4 hr to 50% of the initial value. Conductance averaged 1.4 mS/cm2. Transepithelial potential difference (PD) was usually lumen negative and averaged 0.7 mV. Unidirectional permeability coefficients for 36Cl, 22Na, and radiomannitol were symmetric, proportional to conductance, and followed a rank order compatible with unrestricted passive diffusion. Exposure of the bladder lumen to urine from animals fed saccharin-free or Na saccharin diet hyperpolarized the transepithelial PD by more than 5 mV and raised conductance nearly threefold. Permeability coefficients remained symmetric and compatible with passive diffusion. Exposure of the lumen to solutions with the K+, Na+, and Cl concentrations and osmolality of urine simulated the conductance and PD effects of urine. We conclude that Na saccharin feeding or urine with saccharin does not uniquely affect the permeability of the excised preparation. Small hydrophilic solutes appear to cross the bladder epithelium through paracellular channels which increase in aggregate area during exposure of the lumen to urine. The hyperpolarization induced by lumenal urine is the consequence of the transepithelial K+ gradient.     

Effects of BHA and related phenols on the forestomach of rats

To determine the pathogenesis of BHA-induced forestomach lesions, the nature and time course of the early lesions in the forestomach of Wistar rats were studied. The rats were given BHA at a dose level of 2% in a powdered diet or by oral intubation of 1 g BHA/kg body weight/day in arachis oil. The hyperplastic changes in the mucosa were visible 1 day after the second application. The localization was dependent on the mode of application. Dietary exposure yielded changes in the area of the limiting ridge; oral intubation of BHA produced lesions in the apex of the forestomach. In a subchronic 90-day feeding study in rats, no recognizable effect was observed when 0.125% BHA was incorporated into the diet as a solution in arachis oil. In reversibility studies, severe forestomach lesions observed after feeding 2% BHA for 6, 12 or 15 months regressed almost completely following withdrawal of the BHA for a period of 7 months. BHA induced similar forestomach damage in NMRI mice and Syrian golden hamsters, whereas guinea-pigs, a species having no forestomach, did not show comparable lesions. Substances with similar chemical structure were tested in short-term feeding studies (tert-butylhydroquinone, 4-methoxyphenol, 1,4-dimethoxybenzene, hydroquinone, 3-methoxyphenol, 2-methoxyphenol, anisole, p-cresol, phenol and BHT). Only 4-methoxyphenol strongly affected the forestomach mucosa in a manner similar to that associated with BHA. The methoxy group in the para position seems to be important for the hyperplasiogenic activity.     

亚硝酸钠对人肝癌细胞增殖与凋亡的影响

目的探讨亚硝酸盐对人肝癌细胞增殖和凋亡作用。方法用不同浓度的亚硝酸钠处理人肝癌SMMC-7721细胞不同时间,用MTT分析细胞增殖,荧光显微镜下观察细胞分裂指数和凋亡率,RT-PCR分析c-myc mRNA变化,Westernblot分析缺氧诱导因子-1α(HIF-1α)蛋白表达。结果亚硝酸钠诱导SMMC-7721细胞增殖和凋亡呈双相剂量效应,亚硝酸钠在低浓度时(20～200 mg.L-1),刺激细胞增殖,抑制细胞凋亡,在高浓度时(>800 mg.L-1),抑制增殖,促进凋亡。20～200 mg.L-1亚硝酸钠处理细胞可以明显增加c-myc mRNA和HIF-1α蛋白表达。结论亚硝酸钠在一定浓度范围内刺激SMMC-7721细胞增殖,但是超过一定剂量则诱导细胞凋亡。     

不同葡萄糖浓度对体外培养人牙龈成纤维细胞增殖的影响

目的探讨不同葡萄糖浓度下人牙龈成纤维细胞的生长情况。方法采用体外模型,观察在不同葡萄糖浓度(5.5,7,8.8,10,15及20 mmol/L)细胞培养液中人牙龈成纤维细胞在24 h、3 d、7 d时细胞形态及生长增殖情况。结果当葡萄糖浓度低于8.8 mmol/L时,细胞形态规则,对照组、A组、B组及C组间细胞增殖无明显差异;当浓度高于10 mmol/L时,D组、E组及F组细胞形态呈现不规则,各组增殖能力明显受到抑制。结论高浓度的葡萄糖能够影响人牙龈成纤维细胞的形态,并可抑制其增殖。     

Regression of methyl bromide-induced forestomach lesions in the rat

There is continuing concern about the role of irritation in cancer development. Methyl bromide, a widely used fumigant and known irritant reported to cause forestomach carcinomas in rats, was dissolved in peanut oil and given by gavage at 50 mg/kg body wt to Wistar rats five times per week for 13 to 25 weeks. Starting at Week 13, methyl bromide administration was discontinued for half of the methyl bromide-treated rats (stop treatment group). After that, rats from both the continuous treatment and stop treatment groups were terminated at 4-week intervals to follow the progression of the stomach lesions. Forestomach lesions were not found in control rats receiving peanut oil and killed at 13 or 25 weeks. At 13 weeks the forestomachs from rats receiving methyl bromide were contracted and adherent to the liver and spleen. Inflammation, acanthosis, fibrosis, and a high incidence of pseudoepitheliomatous hyperplasia were found microscopically in treated animals. At 25 weeks, 100% of the rats receiving methyl bromide continuously had hyperplastic lesions of the forestomach which were more severe than those at 13 weeks. Evidence of malignancy was seen in one rat and the lesion was considered a very early carcinoma. In the stop treatment group that received methyl bromide for 13 weeks, there was regression of the stomach lesions, but at the 12-week final sacrifice, adhesions, fibrosis, and mild acanthosis remained. This study illustrates the need for regression experiments for complex forestomach lesions in rodents, especially when an irritating chemical is given by gavage.