L. Aftergood

Effect of varying levels of dietary protein on tumor development and lipid metabolism in rats exposed to aflatoxin.

Reports in the literature concerning the relationship of protein nutrition to aflatoxicosis are contradictory. In an attempt to elucidate this relationship more clearly, we have examined the effects of low, normal, and high protein-containing diets on tumor incidence and development, as well as on several biochemical indices, in rats which have been exposed to low levels of aflatoxin in a “chronic” rather than “acute” situation. In our study, male weanling rats were place for 3 months on otherwise adequate diets containing either 8, 22, or 30% casein with and without aflatoxin B1 at 1.7 ppm. Half of the animals in each group received diets which were further supplemented with the amino acid, cystine, at 0.6% of the diet. (Sulfur-containing amino acids are the most limiting amino acids in casein, and the addition of cystine to the diet serves to improve the biological quality of the protein source.) After 3 months the animals were fed control diets without aflatoxin until they were killed at 1 year. Weight gain was markedly decreased and liver weights increased in response to aflatoxin in all groups except those on the low protein diets, where aflatoxin had no effect on these protein diets, where aflatoxin had no effect on these indices. No tumors were found in the livers of rats fed the low protein, aflatoxin-supplemented diet. In the other groups, the severity of the liver involvement increased progressively with increased protein levels in the diet. When cystine was included in the diet, tumors were observed also in the animals fed the low protein diet; furthermore, the livers of those animals on “normal” and high protein diets were much more severely involved than were the livers of animals on non-cystine supplemented diets. Plasma cholesterol levels were increased in response to aflatoxin when the diets containing 22 and 30% protein were fed and when cystine was included in the 8% protein diet. Liver cholesterol levels were increased in response to aflatoxin in all groups except in those receiving the low protein diets. Among these latter animals, aflatoxin administration had no effect on liver cholesterol values. Changes as a result of aflatoxin administration were also observed in the fatty acid composition of sterol esters, triglycerides, and phospholipids of liver and tumor tissue.

Dietary fat composition and tocopherol requirement: III. Quantitative studies on the relationship between dietary linoleate and vitamin E

The present study has involved biologically titrating linoleate vs. vitamin E using the male rat as the indicator. In the first of the titration studies, the dietary tocopherol level was held constant, while in the second study the linoleate intake was held constant. The investigation was conducted with male rats since these have a much higher linoleate requirement than females. By first depleting such animals of their stores of essential fatty acids by feeding a fat-free diet from weaning, a sensitive test organism was provided. These animals have an immediate need for linoleate during the repletion periods. If an imbalance between linoleate and vitamin E content existed in any of the dietary regimens, such an imbalance would have been more likely noted in test animals actively metabolizing the ingested linoleate. Based upon various nutritional and biochemical indices, the amount of tocopherol ordinarily included in the basic diets fed to our rats, 0.01% as dl-alphatocopheryl acetate, was adequate even when the diet provided up to 5% linoleate; an amount corresponding to ca. 12% of the total calories and providing a ratio of linoleate to the tocopherol of ca. 500:1. In the reverse biological titration with all test diets now providing the constant level of 5% linoleate, ratios of linoleate to vitamin E were satisfactory even in a ratio of as much as 2500:1 (or 0.4 mg gram of vitamin E per polyunsaturated fatty acid). The control animals continued on the fat-free diet indicated that there is a need for added tocopherols even in the absence of linoleate according to a number of biochemical indices. Based upon a number of accepted bioanalytical approaches, the minimum requirement for linoleate by the fat-depleted male rat was found to be between 100–200 mg/day or ca. 1–2% of the caloric intake. Although the fatty acid composition of tissue lipid fractions is markedly affected by the amount of linoleate in the diet, dietary tocopherol supplements have little effect on these values.

Dietary factors and aflatoxin toxicity: I. comparison of the effect of two diets supplemented with aflatoxin B1 upon two different strains of rats

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 B1 included 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.

Dietary fat composition and tocopherol requirement: IV. Safety of polyunsaturated fats

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.

Dietary fat composition and tocopherol requirement: II. Nutritional status of heated and unheated vegetable oils of different ratios of unsaturated fatty acids and vitamin E.

In studies conducted on male and female rats and involving evaluation of growth, reproductive and lactation performances and of lipid peroxidation, no evidence could be found for the need for added vitamin E (a-tocopherol) over and above that naturally present as tocopherols in the vegetable oils investigated. These oils are in common usage in industry, i.e., liquid nonhydrogenated cottonseed oil, a lightly hydrogenated cottonseed oil and a hydrogenated soybean oil shortening. The ratio of polyunsaturates to total tocopherol in the test oils varied from 640:1 to 9:1. Even those oils obtained from a commercial frying operation after a steady state had been attained contained sufficient vitamin E to meet dietary requirements. Results of in vitro peroxide hemolysis tests conducted on the red blood cells of the test animals did not correlate well with biological performance.

Studies of long term administration of aflatoxin to rats as a natural food contaminant.

The effect of feeding aflatoxin, as a natural food contaminant, to rats over long periods of time was studied using multigeneration and longevity tests. The test animals in the multigeneration study consisted of three groups of rats fed diets containing 0, 1 and 10 ppb of aflatoxin (predominantly B1) continued over four generations, with animals of the first and fourth generation fed the diets for 104 weeks. These diets were in proper nutritional balance and included 35% ground roasted peanut products; the ration with 0 ppb aflatoxin excluded the peanuts usually discarded; the one with 1 ppb had the roasted discards returned, while the ration with 10 ppb included the discards in amount 10 times that which had been initially removed. Another longevity study was also performed in which rats were fed diets containing aflatoxin at a level of 80 ppb. In this case, the test peanuts, also fed as a simulated peanut butter at 35% concentration, consisted entirely of roasted peanut discards. Control diets provided no peanut components. Animals fed the low levels of aflatoxin grew as well and actually had a higher percentage survival at 104 weeks than did the animals on the control, aflatoxinfree diets. Organ weights, liver total lipid and cholesterol levels were comparable in all groups. Pathological abnormalities, e.g., hemorrhagic and opaque spots and mottling in some of the livers, were attributed to the aging process since the abnormalities appeared in the control as well as the experimental groups. In the animals fed the aflatoxin at 80 ppb, which has been reported by several investigators to produce well-defined hepatomas in rats, there was liver involvement and some biochemical changes occurred that were not noted in the controls. However, no hepatomas were observed in these animals even after 21 months on this diet. The liver lesions, indicative of a toxic effect, have not been associated with the development of hepatomas. It is possible that some components of the diet used in these experiments may have protected the animal against hepatoma formation. Our studies indicate that there may be a tolerance for aflatoxin as judged by results in one species of rats when whole ground roasted peanuts provide the natural contaminant.

Nutritional evaluation of inter-esterified fats.

Studies with inter-esterified fats prepared to maintain a high level of linoleic acid content have been undertaken in several series of experiments with rats. These fats are as digestible as the liquid nonhydrogenated oils and the biological value of the linoleic acid is not impaired by the inter-esterification. Investigations involving growth, reproduction and lactation, longevity, tissue cholesterol levels and histological tissue examination have revealed that these inter-esterified fats are utilized by the animal similarly to cottonseed oil. No tissue pathology or interferences with any of the nutritional indices are observed. When the inter-esterified fats are included in atherogenic diets, the atheroselerotic lesions which develop in the coronary arteries and aorta of the animals are similar to, but less marked than, those found when animals are fed cholesterol-containing diets with butter oil or conventional margarine oil of the all-hydrogenated type. It is concluded that these inter-esterified fats are at least nutritionally equal to other similar edible fats of equivalent essential fatty acid content.

Effect of dietary fat upon aflatoxicosis in rats fed torula yeast containing diet

This study was designed to study the possible interrelationships between Torula yeast, vitamin E, and the dietary fat source on aflatoxin-induced tumors. Rats were fed Torula yeast-containing basal diets which included 1.7 ppm aflatoxin B1 with either lard, corn oil or no fat, and with or without vitamin E supplements for 3 months. Thereafter, the respective diets without aflatoxin were fed for ca. 9 months. Animals receiving the vitamin E-deficient diets had a high mortality. Although the vitamin E-deficient, aflatoxin-treated rats had lower wt gains than did the vitamin E-deficient controls, they lived twice as long. In addition, regardless of the dietary fat source, the kidneys and adrenals of these vitamin E-deficient, aflatoxin-supplemented rats were found to be significantly heavier than the controls, and plasma cholesterol levels were elevated. Increased amounts of liver lipid were observed in response to aflatoxin in both corn oil-fed and fat-deficient rats. No such differences were observed in the responses of the vitamin E-supplemented groups to aflatoxin. On the corn oil diet, aflatoxin administration resulted in an increased deposition of polyunsaturated fatty acids in cholesteryl ester and phospholipid fractions in livers of vitamin E-deficient rats and the phospholipid fraction of vitamin E-sufficient rats. The vitamin E-deficient rats exhibited necrosis of the liver, which was alleviated when aflatoxin was included in the diet, and calcification of the kidneys, which was potentiated by the dietary aflatoxin. No tumors were observed in these animals. In animals maintained on vitamin E-sufficient diets for 1 year, growth was depressed as a result of aflatoxin administration with the greatest depression occurring in the group fed corn oil. Spleen wt were decreased in all groups given aflatoxin. However, there were no changes in either plasma or liver cholesterol or total liver lipids which could be attributed to aflatoxin administration. When aflatoxin was fed with lard, the cholesteryl ester, triglyceride, and free fatty acid fractions of plasma had decreased amounts of the C20:4 acid. In the cholesteryl ester fraction only, this change was accompanied by increased levels of C16:0, C18:0, and C18:1 acids. In the liver phospholipids, there were increased levels of mono- and polyunsaturated fatty acids and decreases in the saturated fatty acids. All of the animals receiving aflatoxin exhibited severe necrosis and tumor formation in the kidneys; the animals fed lard had the highest level of involvement and those in the fat-free group the least. Liver pathology was the least marked among the rats fed the fat-free diet. Since aflatoxin-induced tumors are rich in lipids, the fat-free diet may be protective to the animal.

Dietary factors and aflatoxin toxicity. II. Effect of fat source upon aflatoxicosis in rats.

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.

Effect of sterculic acid upon aflatoxicosis in rats fed diets containing saturated and unsaturated fat

Investigations on trout have shown that the cyclopropenoid fatty acids, which occur naturally in small amounts in unrefined cottonseed oil, may act as powerful cocarcinogens when fed in conjunction with aflatoxin. Attempts at confirming these findings in mammals, i.e. rats, have been inconclusive. In this study, the effects of sterculic acid and aflatoxin upon lipid metabolism and tumor formation in male rats have been examined using basal diets containing either saturated or unsaturated fat to which the following additions were made: (A) basal diet (no supplements); (B) aflatoxin B1 at 1.7 ppm; (C) sterculic acid at 210 ppm; and (D) aflatoxin B1 at 1.7 ppm, plus sterculic acid at 210 ppm. The rats consumed these diets for 3 months and, thereafter, were fed the unsupplemented basal diet until sacrifice 9 months later. Growth was depressed in rats in groups B, C, and D, but no synergistic inhibition was observed, regardless of the fat source. Liver wt doubled in response to aflatoxin; however, only when the diet contained unsaturated fat did sterculic acid, in combination with aflatoxin, exaggerate the increase in liver wt (a reflection of the more severe liver pathology observed in these rats). In the animals fed the saturated fat diet, aflatoxin administration to animals fed the control or sterculic acid supplemented diets resulted in marked increases in plasma cholesterol levels; the unsaturated fat diets, supplemented with aflatoxin, evoked a slight increase in plasma cholesterol content which was nullified by sterculic acid supplementation.