D.B. Clayson

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.

Effects of deoxynivalenol (vomitoxin) on the humoral and cellular immunity of mice.

Sublethal doses (0.00, 0.25, 0.50 and 1.00 mg/kg b.w./day) of vomitoxin (deoxynivalenol; DON) were studied for their effects on humoral and cellular immunity and serum proteins of inbred, male Swiss Webster mice in a series of 4 separate experiments. Vomitoxin was added to basal diet (less than the detection limit, i.e., less than 0.05 micrograms of vomitoxin per g of feed) and administered to mice for 5 weeks beginning at 21 days of age. Mice in experiment 2 were fed the basal diet for 40 days in addition to the 5-week treatment with vomitoxin. The 1.00 mg/kg dose of vomitoxin resulted in a statistically significant reduction in the serum levels of alpha 1 and alpha 2-globulins, an increase in total serum albumin, and a reduction in feed consumption and body weight gain compared to the control group. The 0.50 mg/kg dose of vomitoxin resulted in significantly reduced serum levels of alpha 2- and beta-globulins while a significant reduction of feed consumption was evident only during Week 4. Similarly, body weight gain in this group of mice was significantly reduced during Week 2 but increased to normal levels during Week 3 and remained parallel to the control for Week 4 and 5. Both levels (0.50 and 1.00 mg/kg) of vomitoxin resulted in a reduced, dose-related, time-to-death interval following a challenge with L. monocytogenes and increased proliferative capacity of splenic lymphocyte cultures stimulated with the phytohemagglutinin P (PHA-P) mitogen compared to the control group of mice. The 0.25 mg/kg dose of vomitoxin did not have any significant effects on the parameters studied. A reasonable estimation of a no effect level for immunologic effects in mice based on these and previous immunological studies would seem to be between 0.25 and 0.50 mg/kg b.w./day.

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.

Short-term pathological and proliferative effects of butylated hydroxyanisole and other phenolic anti-oxidants in the forestomach of Fischer 344 rats

Food grade butylated hydroxyanisole (BHA) when incorporated in the diet and fed to male Fischer 344 rats for 9 or 27 days induced proliferative squamous epithelial changes in the lesser curvature of the forestomach proximate to the glandular stomach. These changes were assessed histopathologically and by [methyl-3H]thymidine radioautography. It was shown that BHA mixed dry into powdered diet, incorporated into the diet in corn oil, or in a pelleted diet, induced similar effects. When levels of 2%, 1%, 0.5%, 0.25%, 0.1% and 0% BHA were incorporated in rat diet for 9 days, the proliferative effect appeared to show a no effect level at 0.25% based on the [methyl-3H]thymidine-labelling index. Other food use antioxidants, namely butylated hydroxytoluene or tertiary butylhydroquinone, induced a lesser response than BHA at the maximum dose employed in the study. Propyl gallate was without effect. Propyl-4-hydroxybenzoate, a food use phenol, on the other hand, induced a less pronounced response than BHA but was more effective than the other antioxidants. Because increased cellular proliferation often provides an optimal milieu for tumor formation, it is suggested that these observations may be relevant to rat forestomach tumors induced by BHA.

Histopathological and radioautographical studies on the forestomach of F344 rats treated with butylated hydroxyanisole and related chemicals.

Butylated hydroxyanisole, when fed to male Fischer 344 rats for periods of 9 days or more, led to forestomach epithelial cell necrosis and regeneration. Both the induced proliferation and the histopathological changes were considerably more prevalent in the prefundic region of the forestomach than in the mid-region. Using [Me-3H]thymidine, a specific DNA precursor, and radioautography it was shown that the effect of the antioxidant was apparently threshold at 0.25% in the diet after both 9 days and 3 months of treatment and that the proliferation was dependent on the continuous presence of the antioxidant in the diet at 3 and 6 months. Some other phenols and acids were investigated after 9-27 days feeding; most induced some degree of epithelial cell proliferation in the rat forestomach, although some had a greater effect on the mid-region than on the prefundic region. These observations are discussed in terms of the likely relevance of butylated hydroxyanisole-induced forestomach tumours to the possibility that the antioxidant may lead to cancer in humans exposed to lower levels in their diet.

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.

ICPEMC publication no. 13 the need for biological risk assessment in reaching decisions about carcinogens (MTR07214)

The prudent assumption that carcinogen bioassays in rodents predict for human carcinogenicity is examined. It is suggested that in certain cases, as for example the induction of tumors against a high incidence in controls, or in situations in which high dose toxicity may be a critical factor in the induction of cancer, the probability that animal bioassays predict for humans may be low. The term biological risk assessment is introduced to describe that part of risk assessment concerned with the relevance of specific animal results to the induction of human cancer. Biological risk assessment, which is almost entirely dependent on an understanding of carcinogenesis mechanisms, is an important addition to present mathematical modeling used to predict the effects of animal carcinogens that have been demonstrated after high dose exposure, to the effects of the much smaller doses to which humans are perceived to be exposed. Evidence for the conclusions reached by biological risk assessment may sometimes be supported by a careful review of human epidemiological data.

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.