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Previous studies in this laboratory with rats fed low levels of aflatoxin as a component of peanut discards, suggested either a strain tolerance for aflatoxin or a possible protective factor present in the diets used. In this study, two strains of rats, the Charles-River strain and the former USC strain, were used to test the effect of 1.7 ppm purified aflatoxin B1included for 3 months in two different diets; one previously used in this laboratory and one used by other investigators in aflatoxin studies. After an experimental period of either 12 or 18 months, growth, mortality, gross pathology, and organ wt were measured, and histopathological examination and biochemical analyses were performed. Plasma and liver cholesterol levels, total liver lipids, and fatty acids in the various lipid fractions of plasma, liver, and liver tumor lipids were measured. Both strains of rats proved to be susceptible to aflatoxin toxicity at this level as manifested by the appearance of hepatomas; however, liver involvement was more extensive in the Charles-River rats. The diet used by other investigators produced symptoms similar to those observed as a result of essential fatty acid deficiency and also affected the response to aflatoxin through an aggravation of symptoms, i.e. an inhibition of growth and increased size and severity of the liver tumors.

Previous studies in this laboratory have indicated that the tumorigenic and biochemical response of rats to aflatoxin may be affected by diet. To clarify the possible importance of the fat component of the diet in altering these biochemical responses a cross-over experiment was devised in which peanut oil and lard were reversed in two diets containing different protein sources and vitamin mixtures. It was found that more concentrated doses of aflatoxin administered for a shorter period of time had a more inhibitory effect upon growth, and liver pathology also appeared to be worse than when smaller doses were given for longer periods of time. There were slight differences in pathology due to the basal diet among peanut oilfed rats, and large differences when the fat component was lard. Plasma cholesterol levels were elevated as a result of the aflatoxin administration, regardless of the diet, and liver cholesterol levels were elevated in response to aflatoxin when diets containing the more varied protein source were fed. In all fractions of plasma fatty acids, fatty acid patterns similar to those observed in marginal essential fatty acid deficiency were seen when lard was fed. The observations that the more restricted protein diet or some component of it, reacts with lard to produce signs similar to those typical of essential fatty acid deficiency and that this regimen produces the most severe liver pathology further establishes the importance of the diet as a means of modifying the response of the organism to aflatoxin.

Abstract: In a long-term multigeneration study, conducted in our laboratories for 32-years, with occasional longevity and histopathological evaluations included, rats of our own inbred strain (originally of Wistar derivation) were fed semisynthetic diets comprising whole wheat, skim milk powder, and fat in the form of margarine products. The total source of tocopherols was the dietary fat itself. Saturated fatty acid content (S) remained relatively constant at about 20% of the fat and total tocopherol level also remained constant at about 0.12% of the fat. Polyunsaturated fatty acid (P) content, however, progressively increased almost fourfold, from 7.5% to 28.5% and alphatocopherol levels decreased to one-half level, from 0.033% to 0.016% of the fat. Hence, the ratio of polyunsaturated fatty acids to alpha-tocopherol content changed markedly from 227∶1 to 1780∶1, with other factors (relative to fat composition) held constant during the 32-year period of feedings and observations. Fat level in the diet increased over the years from 9.2% to 16.0% or from about 21% to about 33% of the caloric intake. Thus, quality and quantity of the fat in the diet progressively changed, and the impact of these changes was evaluated by comparing biological performances of the successive generations. Growth and reproduction and lactation performances were noted to be regularly satisfactory and comparable from generation to generation throughout the experimental period. Longevity studies conducted on arbitrarily selected generations also provided data showing no deleterious effects associated with a dietary change. Histopathological examinations of tissue revealed minimal myocarditis and no malignant tumors which could be attributed to a dietary factor. No vitamin E deficiencies were observed. Even the in vitro peroxide hemolysis values for the red blood cells of the animals, fed the diets containing the higher levels of polyunsaturated fatty acids, were low, indicating that the dietary fats provided sufficient absorbable tocopherol to protect the potentially oxidizable unsaturated fatty acids in the erythrocyte membrane. Biochemical data reflected responses to aging and not to any specific diet fed. It is concluded that a diet providing as much as 33% of the calories as a fat, the latter containing up to 28.5% polyunsaturated fatty acids, substantially of the essential fatty acid type, with a P/S ratio of up to 1.6∶1 and a polyunsaturated fatty acid to alphatocopherol ratio as high as 1780∶1] produces no undesirable effects in the rat.

A blend of hexitols, comprising 80 parts by weight of mannitol and 20 parts of sorbitol, has been used in place of a frying fat in roasting nuts. Even though physicochemical tests demonstrated no measurable deterioration of the hexitol components on continuing repetitive use, biological studies were carried out to establish the safety of the end product. The latter was the novel dry roasted peanut product; the control consisted of conventionally air roasted peanuts. Both products were ground to peanut butter consistency and then stabilized with added hydrogenated peanut oil to facilitate the feeding programs. Male and female rats were maintained on the two peanut products for four generations. It was found that rats subsisting on diets containing the regular peanut butter increased in weight at a somewhat greater rate than did those on the diet containing the special peanut product. Efficiency of food utilization was not adversely affected and overall weight gains were satisfactory. All rats appeared normal and healthy throughout the experiment. There was no morbidity and no diarrhea. Breeding performance was slightly better among those rats on the special peanut product diet. No pathology was observed at autopsy and there were no differences in organ weights among the rats on the two diets. Total liver lipid and liver cholesterol levels were somewhat higher among the control peanut butter-fed rats, as compared to those on the “special” peanut butter diet. On the basis of (a) published related studies involving biological evaluations of the control peanut butter diet vs. the same adequate diet but containing no peanut components and (b) the present findings, it is concluded that no unknown factors of a toxic nature are present in peanuts roasted in the molten hexitol blend.

In general, the native tocopherols in polyunsaturated vegetable oils such as cottonseed oil, corn oil and their lightly hydrogenated products include sufficient vitamin E for growth, reproduction, lactation and normal lipid metabolism in the rat. The administration of vitamin E to animals fed diets deficient in essential fatty acids (e.g., a hydrogenated coconut oil or a fat-free diet) does not stimulate growth or reproductive performance per se, although testes development in the male rats is improved and some improvement in lipid metabolism is also noted. Hemolysis of the erythrocytes in vitro by hydrogen peroxide is increased in animals on diets rich (30%) in polyunsaturated vegetable oils or on diets providing no essential fatty acids at all. However, the conditions of the in vitro hemolysis test are not related to those in vivo and the in vitro test is not a measure of erythrocyte fragility. In addition, the in vitro hemolysis test does not necessarily reflect plasma tocopherol levels nor an abnormal nutritional state as a result of subsistence on high linoleate, low tocopherol intake, but rather measures the susceptibility to oxidation of a labile biological substrate and indicates the effective balance between potentially oxidizable lipids (polyunsaturates) in the stroma of the red blood cell and the antioxidant present (tocopherol or vitamin E). The labile lipid substrate may be either of exogenous origin (diet) or may be formed endogenously through tissue synthesis (as a result of an essential fatty acid deficiency). It is concluded that the in vitro hemolysis test may not be a valid indicator of vitamin E nutriture unless it is used in conjunction with other nutritional tests.