E. Lok

Calorie restriction and cellular proliferation in various tissues of the female Swiss Webster mouse

Two experiments have been conducted to determine the effects of calorie restriction on cellular proliferation in female mouse tissues. In the first experiment, 25% calorie restriction led to a decrease in cellular proliferation, measured by the [3H]thymidine labeling index, in each of the 7 tissues examined. The duct lining cells of the mammary gland were the most affected. This conclusion was confirmed in the second experiment in which levels of 0, 10, 20, 30 and 40% calorie restriction were employed. The difference in response of the duct cells of the mammary gland and the crypt cells of the colo-rectum is discussed in light of the opinion that the formation of cancer of these tissues in humans may be markedly affected by diet.

Domoic acid poisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract.

Consumption of cultivated blue mussels from Prince Edward Island was recently associated with episodes of gastro-intestinal and neurological distress. Extracts of the toxic mussels, tested in the mouse bioassay for paralytic shellfish poison, caused an atypical response characterized by scratching, convulsions and death. The present investigation shows that the domoic acid present in toxic mussels can produce in mice and rats signs identical to those induced by mussel extracts. These studies, preliminary in nature by virtue of the scarcity of domoic acid, gave ip no-effect levels in mice of 0.59 mg/kg body weight based on the behavioural response (scratching) and 2.4 mg/kg for death. These levels correspond to levels of 24 and 94 ppm in mussels. When administered orally doses of between 35 and 70 mg domoic acid/kg body weight were required to produce toxicity in mice and rats. This reduced toxicity is consistent with a lack of absorption from the gastro-intestinal tract: faecal excretion accounted for 102 +/- 17% and 98 +/- 12% (mean +/- SE) of the domoic acid administered to mice and rats, respectively. Since human intoxication occurred at an estimated 1-5 mg domoic acid/kg body weight, susceptible individuals appear to be more sensitive than rodents to the oral toxicity of domoic acid.

Dietary restriction, cell proliferation and carcinogenesis: a preliminary study.

Four groups of female Swiss Webster mice were given either laboratory chow or a purified (semi-synthetic) diet (AIN-76A) either ad libitum or at 75% of the ad libitum rate for about 30 days. Three tissues, the crypt cells of the jejunum, the dermis and the basal epithelial cells of the esophagus were investigated using [3H]thymidine labelling and by counting mitoses; four other tissues, the alveolar cells of the mammary gland, the crypt cells of the duodenum and colo-rectum, and the transitional cells of the urinary bladder were examined using [3H]thymidine labelling only. In each case dietary restriction led to a reduction of cellular proliferation assessed by these indices. The potential of the approach for the study of the effects of dietary modification on the induction of cancer is discussed.

A carcinogenesis reversibility study of the effects of butylated hydroxyanisole on the forestomach and urinary bladder in male fischer 344 rats

A reversibility study was initiated to determine if the length of feeding with 2% butylated hydroxyanisole (BHA) altered the incidence of forestomach lesions observed after a 24-month observation period. Groups of male Fischer 344 rats were fed 2% BHA for 0, 3, 6, 12, and 24 months and then the basal diet for the completion of the 24-month experimental period. Subgroups were serially sacrificed for histopathological examination and [methyl-3H]thymidine radioautography at the time when each group of animals was transferred to the basal diet and also at 15 months. The results showed that except for carcinomas and some epithelial downgrowths, cellular proliferation, measured by radioautography in the epithelium lining the greater and the lesser curvature of the forestomach, remained dependent on the continuous presence of 2% BHA for, at least, 12 months. Superficial hyperplasias, inflammatory lesions and many of the papillomas regressed after cessation of treatment at 12 months. The epithelial downgrowths did not appear to enlarge after the BHA was withdrawn. The squamous cell carcinomas occurred in almost identical yields whether the rats were fed 2% BHA for 12 months and then returned to the basal diet for 12 months or received 2% BHA continuously for 24 months. It is shown here that at several times, 2% BHA stimulated the [methyl-3H]thymidine labelling index of the transitional epithelium of the urinary bladder and that at 3 months the no observed effect level was greater than 0.5% BHA. The significance of the studies on the forestomach and bladder epithelia are discussed. It is concluded that the lesions induced by BHA are most unlikely to be relevant to humans exposed to much lower levels of BHA.

A 13-week feeding study of butylated hydroxyanisole: The subsequent regression of the induced lesions in male fischer 344 rat forestomach epithelium

Feeding butylated hydroxyanisole (BHA) to male Fischer 344 rats at concentrations of 2, 0.5, 0.25, 0.1 and 0% for 13 weeks led to proliferative lesions developing in the forestomach epithelium of the 2%-treated rats but not in other groups. The [methyl-3H]thymidine labelling index was raised in the 2%- and 0.5%-treated groups and showed an apparent no observable effect level at 0.25%. Within 1 week after withdrawal of BHA the labelling indexes in all treated groups returned to near the values in the controls. The induced mucosal lesions, however, reverted more slowly and even after 9 weeks on the basal diet, the stratified squamous epithelium along the lesser curvature, was still slightly thicker than the control. There were multilayered basal cell processes in the lamina propria with connections to the basal cell layer. The possible significance of these results to the ultimate development of cancer is discussed.

Short-term effects of various phenols and acids on the fischer 344 male rat forestomach epithelium

A series of phenols and acids was fed to rats for 9 days to determine effects on the [methyl-3H]thymidine labelling index and the histological appearance of the forestomach. A variety of proliferative effects in the rat forestomach were observed which paralleled changes in the [methyl-3H] thymidine labelling index. The 3-tert-butyl isomer of butylated hydroxyanisole (BHA) was as effective as the food grade mixture. In the 4-hydroxybenzoic acid ester series, activity was not found either with the free acid or the methyl ester. With the ethyl, n-propyl and n-butyl esters activity in the perfundic region of the forestomach increased with alkyl chain length, the n-butyl ester being nearly as effective as BHA. In contrast, 4-methoxyyphenol and propionic acid demonstrated their greatest effects in the midregion of the forestomach, the action of propionic acid not being apparent until 21 or 27 days of treatment. It was also found that after a 9-day treatment involving coadministration of BHA and acetylsalicyclic acid, the overall effect of the antioxidant on the forestomach was greatly reduced.

An 85-day study of butylated hydroxyanisole in the cynomolgus monkey

Groups of 8 cynomolgus monkeys (Macaca fascicularis) were given 500, 125 and 0 mg/kg body wt butylated hydroxyanisole (BHA) by gavage in corn oil 5 times/week for 20 days, after which the high dose was halved. No significant adverse clinical signs nor abnormal fibroscopic observations were noted before the experiment was terminated at 85 days. Blood levels of glucose, albumin, chloride, red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT) and mean corpuscular hemoglobin (MCH) were altered in a dose related manner but the altered values were well within normal ranges reported for this species. While histopathology showed no treatment related effects, the mitotic index was elevated 1.9-fold in the distal esophagus of monkeys in the high but not in the low dose group. Liver weights were increased in a dose related manner but liver monooxygenases, with the exception of decreased ethoxyresorufin deethylase activity, were all within normal limits. BHA given orally to monkeys at about the maximum tolerated dose failed to induce the massive changes noted with rats given 2% dietary BHA.

The potential for the use of cell proliferation studies in carcinogen risk assessment.

The use of observations on cellular proliferation in assessing the mechanism of action of certain factors in the genesis of tumors of the urinary bladder, forestomach, and intestine, or on the effects of dietary restriction, is illustrated. It is suggested that, particularly with nongenotoxic carcinogens, such studies may be of great use in risk assessment especially for those cases in which animals are exposed at very much higher levels of the test agent in the carcinogenesis bioassay than are humans as the result of the environmental or other use of the agent.

The Importance of Cellular Proliferation Induced by BHA and BHT

Phenolic antioxidants have been used for many years to prevent oxidative spoilage of food (rancidity). They are of major importance in protecting foodstuffs during transportation and storage. Antioxidants thus constitute an essential element in the modem centralized food processing and distribution industries. In Canada, only three phenolic antioxidants are permitted to be added to food: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG). The mean dietary intake of BHA and BHT, as a result of their useas food additives, is relatively low; in Canada the intake overall ages is 7.40 mg/person/day for bothBHA and BHT, leading to a combined totalof 0.26 mg/kg/day (Kirkpatrick andLauer, 1986).

Short-term effects of butylated hydroxytoluene on the Wistar rat liver, urinary bladder and thyroid gland

Long-term feeding of butylated hydroxytoluene (BHT) to rats and mice has been linked to the enhancement of the incidence of liver tumors. It is shown in this paper that in the liver, urinary bladder and thyroid of the male Wistar rat, feeding the highest tolerated doses of BHT for 30 days does not lead to detectable increases in [3H]thymidine labeling. On the other hand, treatment of rats with 0.5% dietary BHT leads to a time-limited increase in liver cell [3H]thymidine labeling that subsided to control values within 8 days. This increase in [3H]thymidine labeling in the liver is accompanied by an unexpectedly large increase in the mitotic index. These results are discussed in the light of the behavior of certain rodent liver tumorigens.