Effects of a dietary load of acid or base on changes induced by lactose in rats.

Feeding lactose or other slowly digestible carbohydrates to adult mammals may induce a variety of effects including hyperplasia and neoplasia. The most fundamental effect probably is the increased production in the large intestine of short-chain fatty acids (SCFA) resulting from increased fermentation of carbohydrate residues. To find out whether the increased production of these acidic compounds is involved in the induction of certain alterations caused by low-digestibility carbohydrates, the modifying effects of an acidifying (NH4Cl) or an alkalizing (KHCO3) diet supplement on lactose-induced changes in rats were studied. Three groups of 50 rats per sex were fed a 20% lactose diet unsupplemented or supplemented with 1% NH4Cl or 2% KHCO3, for at most 2.5 yr. One control group was fed the basal diet which contained wheat starch instead of lactose. Feeding lactose resulted in wet faecal pellets, reduced pH of the faeces, higher intake of food and water, lower body weights, increased caecal weights and fewer deaths. These effects were not significantly modified by NH4Cl or KHCO3. Feeding lactose increased urinary calcium levels, the effect being enhanced by NH4Cl and reduced by KHCO3. Lactose also tended to increase blood values of alkaline phosphatase and to decrease those for bicarbonate and base excess. These tendencies were generally more marked with NH4Cl, and less marked or absent with KHCO3. In addition, rats fed lactose showed decreased severity of nephrosis, increased mineralization and hyperplasia of the renal pelvic epithelium, and relatively high incidences of Leydig cell hyperplasia and neoplasia. NH4Cl supplementation was associated with a relatively small number of single and multiple tumours, with decreased incidences of hyperplasia and mineralization of the renal pelvis epithelium and with a markedly reduced incidence of proliferative changes in the adrenal medulla. With the KHCO3 supplement the incidences of Leydig cell proliferation and of bladder tumours were relatively high. These findings, in particular the differences between the diet groups in urinary calcium levels and possibly also the variations in blood levels of alkaline phosphatase, bicarbonate and base excess, suggest that the acidic end products of carbohydrate fermentation (SCFA) act as an acid load on the body.

Comparison of cereal-based diet with purified diet by short-term feeding studies in rats, mice and hamsters, with emphasis on toxicity characteristics.

Animal diets used in toxicity studies are prepared either from natural ingredients (cereal-based diet) or from more refined products (purified diet). The type of diet may influence both the outcome of the study and the values obtained with the various parameters in test and control animals. To detect the parameters sensitive to changes in diet composition, short-term (4-wk) studies were conducted in rats, mice and hamsters fed either a cereal-based diet or the AIN-76A purified diet supplemented with vitamins and minerals at the highest recommended levels for each of the species used. Although the purified diet was more palatable to rats and showed a higher protein quality, growth rate and food intake were generally slightly higher with the cereal-based diet in each of the species examined. The haematological values of the two diet groups were generally comparable. On the cereal-based diet the production of faeces was considerably higher than on the purified diet and was accompanied by a higher weight of the caecum. These findings were attributed to the relatively high level and mixed composition of the fibre fraction in the cereal-based diet. Blood levels of cholesterol and phospholipids were clearly lower on the cereal-based diet than on the purified diet. Because the differences were probably due to the level and composition of the fibre fraction, they support the suggestion to replace the 5% cellulose of the AIN-76A diet by a higher level of a more composite but well defined source of dietary fibre.

Effects of high levels of brussels sprouts in the diet of rats.

To examine the possible harmful effects of feeding Brussels sprouts to rats, groups of 10 male, weanling rats received the non-dehydrated vegetable in moist diets at levels providing 2.5-30% of the dry matter for 4 wk. A first study comprised test diets with 15 and 30% of the dry matter as uncooked or cooked Brussels sprouts and control diets without and with 0.2% potassium thiocyanate (KSCN) for comparison. The second study comprised diets with 0, 2.5, 5, 10 and 20% of the cooked vegetable and diets with 0 and 20% of the cooked vegetable with extra iodine. Diets with the uncooked vegetable contained considerably less intact glucosinolates than did diets with the cooked product, probably as a result of more extensive enzymatic degradation in the uncooked product. Growth depression and decreased food intake, not accompanied by decreased food efficiency, occurred in rats fed 10% or more dry matter as Brussels sprouts. These findings were less marked with the cooked than with the uncooked vegetable, probably because of unpalatability. Decreased levels of blood haemoglobin and plasma thyroxin were found with 15% or more Brussels sprouts. Prothrombin times were increased if 2.5% or more was fed. Thyroid stimulating hormone was increased by feeding potassium thiocyanate, but not by feeding the vegetable. Increased kidney weights and impaired kidney function not accompanied by microscopic renal changes were observed in rats fed 10% or more Brussels sprouts. Increased liver weights, which occurred from the 5% level, were accompanied by microscopic hepatic changes only at feeding levels from 10% of the cooked vegetable. 'Morphological activation' of the thyroid was increased with 10% or more of the cooked vegetable and with 0.2% KSCN. Iodine supplementation of the diets did not influence the results obtained with the vegetable. These studies indicated that 2.5% Brussels sprouts dry matter in the diet was not without effect, and that the thyroid characteristics were less sensitive to Brussels sprouts than were other criteria examined.

Chronic toxicity and carcinogenicity study of isomalt in rats and mice.

The chronic toxicity and possible carcinogenicity of the sugar replacer isomalt was studied in Wistar rats and Swiss mice. Groups of 50 animals of each sex were fed 0, 2.5, 5 or 10% isomalt in the diet for nearly 2.5 yr (rats) or 2 yr (mice). Control groups received either basal diet with 10% maize starch or basal diet with 10% sucrose. Additional groups of ten rats/sex were fed the same diets and were killed after 1 yr. Isomalt and sucrose were included in the diet at the expense of maize starch. Administration of isomalt was started, in rats, in utero, and in mice, at weaning age. Feeding isomalt did not affect the appearance or behaviour of rats or mice, nor did it cause diarrhoea. Mortality rate was unaffected. Body weights of rats and mice fed 10% isomalt were generally slightly lower than those of controls. Periodic examinations of rats for haematological criteria, clinical chemistry of the blood, urine composition and kidney function did not reveal any changes of toxicological significance. Periodic haematological examinations of mice were likewise negative. Caecal enlargement was observed in rats and mice of the high-dose group, but the microscopic structure of the caecal wall was unaffected. An increased number of treated male and female rats showed hyperplasia of the urothelium in the renal pelvis accompanied by mineralization, whereas the number of females showing corticomedullary mineralization was decreased in the treated groups. The incidence, type or location of neoplasia provided no evidence of a carcinogenic potential of isomalt. Feeding 10% sucrose did not induce significant differences compared with the controls fed 10% maize starch, whereas isomalt at levels of up to 10% produced some of the changes that are common to rats fed high levels of poorly digestible carbohydrates.

Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/carcinogenicity testing.

In previous studies we observed an increased incidence of hyperplasia in the epithelium of the urinary bladder of rats fed cereal-based stock diet supplemented with 6% monosodium glutamate (MSG) for 3 months. Hyperplasia was not enhanced, however, when 6% MSG was fed in a purified casein diet. Further studies have been conducted to identify the dietary factor that caused the different response with the two diets. Feeding MSG had a marked alkalizing effect on the urine. Rats fed purified diet produced urine of higher acidity than did those fed stock diet, a finding attributed to the greater excess of base in the stock diet. When diets with a considerable excess of cations were fed, urinary pH showed a characteristic pattern of widely differing values during a 24-hr period, with high values (pH greater than or equal to 8] for several hours of darkness, when food intake was high, declining during the day to a minimum at the end of the light period. Hyperplasia of the bladder epithelium was induced not only by feeding MSG, but also by feeding 5% of the alkalizing salt KHCO3, both in purified diet and in stock diet. The epithelial response to an alkalizing substance was prevented by simultaneous feeding of the acidifying salt NH4Cl. These findings indicate that the bladder changes induced by MSG are attributable to its alkalizing properties rather than to MSG per se. Moderate to severe hyperplasia of the bladder epithelium was induced also by feeding 5% NH4Cl in purified diet, a procedure accompanied by a further lowering of urinary pH. These findings showed that hyperplasia of the bladder epithelium of rats can be induced both by acidifying and by alkalizing the urine through manipulation of the acid-base balance of the basal diet. There is thus a possibility that, in carcinogenicity studies, administration of compounds to rats in the form of a salt may lead to erroneous conclusions.

The effect of pyridoxine on the number of lymphocytes in the blood of rats fed caramel colour (III).

The effect of vitamin E, folic acid, pyridoxine (vitamin B6) and choline on the reduction in circulating lymphocytes in the blood of rats fed Caramel Colour (III) also known as ammonia caramel colour (beer type; AC) has been examined. It was found that the reduction in the number of circulating lymphocytes in rats fed AC could be prevented by the addition of pyridoxine to the diet. The activity of AC in reducing the number of circulating lymphocytes also closely resembled that of the pyridoxine antagonist 4'-deoxypyridoxine. After the isolation and identification of 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI), comparative studies indicated that THI was the component of AC responsible for reducing the number of circulating lymphocytes. Although the effect of AC was reduced or prevented by increasing dietary pyridoxine, the lymphocyte reduction associated with the administration of THI was not materially affected by the dietary level of pyridoxine.

Effects of alkali-treated proteins: feeding studies with free and protein-bound lysinoalanine in rats and other animals.

To find out whether alkali-treated proteins posses nephrotoxic properties, feeding studies were conducted with drastically treated soybean protein and casein, and also with lysinoalanine (LAL), the amino acid known to be formed in protein subjected to high pH at elevated temperature. The feeding of synthesized LAL to rats at dietary levels of 100 ppm and above induced typical renal changes, called nephrocytomegalia. No such changes or any other indications of toxicity were observed, however, upon feeding much higher levels of LAL (up to 6,000 ppm) when provided as the protein-bound compound in alkali-treated casein or soybean protein. When set free by complete acid hydrolysis, LAL induced considerable renal activity, comparable to that of the synthetic compound. These results indicate that alkali treatment of proteins does not induce nephrotoxic properties provided that the compound remains protein-bound. Some nephrotoxic activity was observed, however, with peptide-boound LAL in break-down products (molecular weight less than 5,000) of alkali-treated casein, but considerably less than that of the free compound. LAL-analyses in blood, urine, and feces of rats fed free or protein-bound LAL indicated a positive correlation between intestinal absorption and nephrotoxic potential. No renal changes were encountered upon feeding diets with 1,000 ppm synthetic LAL to mice, hamsters, rabbits, quail, dogs or monkeys, which suggest a species specificity of LAL-induced renal changes in rats.