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A 16-week feeding test of Tower ( Brassica napus) and Span ( Brassica campestris) rapeseed protein concentrates (RPC) was carried out on male and female Charles River CD (Sprague-Dawley) weanling rats. The diets contained 10% or 20% protein (N × 6.25) from each RPC and were supplemented with 150 mg zinc/kg. The control groups received a purified diet with casein or a laboratory chow diet. The results of the test diet groups did not reveal any adverse effect due to RPC on thyroid metabolism or the metabolism of zinc iron, copper, manganese, calcium, phosphorus, and magnesium. Nephrocalcinosis was observed in all female rats that were fed the test diets and the casein control diet, and therefore it could not be attributed to dietary RPC. [ABSTRACT FROM AUTHOR]

Two rapeseed protein concentrates (RPC) supplemented with zinc oxide were fed to male and female rats at two levels providing approximately half or all of the protein in a purified basal diet containing 20% protein ( N×6.25). Control groups were given either a purified basal diet with casein or a laboratory chow diet. After 16 weeks, there were no significant ( P<0.05) differences in body weight except the male rats fed the casein control diet had higher body weights than did other male rats. No significant differences were observed in organ weights including thyroids, hematology, bone marrow differential counts, serum glucose, urea nitrogen, total protein, albumin/globulin ratio, cholesterol, transaminases or alkaline phosphatase. The levels of serum vitamins A and E of females fed some of the RPC diets were less than in those fed the caseincontrol diet, but equal to those in females fed laboratory chow diet. Females fed either RPC or casein in the semi-purified diets, which contained zinc oxide, exhibited kidney calcification. [ABSTRACT FROM AUTHOR]

A total of 80 menstruating rhesus monkeys (Macaca mulatta) were equally and randomly divided among groups receiving 0, 5, 20, 40, or 80 μg of Aroclor 1254/kg body weight/day during a 6-year toxicological-reproduction study. During the first 3 years of the study, 4 of the treated monkeys became moribund and were euthanized; 3 had endometriosis. This finding suggested a possible link between the PCB treatment and the occurrence of endometriosis. However, neither a laparoscopic examination of the control and high-dose monkeys nor the necropsy data provided evidence for a possible link between the PCB treatment and the observed incidence (37% (6/16) of controls; 25% (16/64) of treated monkeys and/or the severity of the endometrial lesions. Additional clinical and historical data not contained in previous reports are presented to facilitate independent evaluation of the relationship between PCB ingestion and endometriosis. We conclude that the incidence and severity of the endometriosis lesions observed in the rhesus monkeys utilized in this study did not have any relationship with the dosages of Aroclor 1254 they ingested. [ABSTRACT FROM AUTHOR]

A Short-Term Feeding Study with Deoxynivalenol (Vomitoxin) Using Rats. ARNOLD, D. L., KARPINSKI, K. F., MCGUIRE, P. F., NERA, E. A., ZAW1DZKA, Z. Z., LOK, E., CAMPBELL, J. S., TRYPHONAS, L., AND SCOTT, P. M. (1986). 6,691–696. Groups of 25 male and 25 female Sprague-Dawley rats were fed diets containing 0, 0.25, 0.5, or 1.0 mg of deoxynivalenol (DON)/kg body wt for approximately 9 weeks. Each animal's body weight and feed consumption were measured weekly. Upon termination of the study, each animal's body, heart, liver, spleen, thymus, and kidneys were weighed. A hematological assessment and a 16-parameter serum evaluation were conducted and 8 animals from each group were randomly selected to receive tritiated thymidine iv to assess mitotic activity in the esophagus, jejunum, and spleen. A statistically significant, dose-related decrease in body weight gain was observed for all treated females, but only the males dosed at 1.0 mg/kg were found to have a treatment-related weight gain suppression. The reduced body weight was attributed to a reduced feed consumption. Reductions that were observed in absolute organ weights, were not apparent after adjusting for body weight suppression. No dose-related hematological findings were found. Serum chemistry changes included increased concentrations of chloride and decreased concentrations of CO and albumin, but only in the females. No histopathological lesions were attributed to DON treatment, but significant decreases in thymidine labeling occurred in the spleens and jejunums from the males dosed at 1.0 mg/kg. [ABSTRACT FROM PUBLISHER]

In previous work we have shown that changing the fatty acid composition of a constant amount of fat in a modified AIN-76A diet affected the level of ductular cell proliferation in the mammary glands of young virgin female Swiss Webster mice. In particular, linoleic acid concentrations of 5-10% of the total fat in the diet led to variable but appreciably higher levels of proliferation than did higher levels of linoleic acid. In this report it is shown that feeding low levels of the total fat as alpha-linolenic acid (0-5%) resulted in a similar effect. In addition the effects of other fats including menhaden oil, were further investigated.

Butylated hydroxytoluene (BHT) is a synthetic, food-use, phenolic antioxidant. It has previously been demonstrated to be operationally non-genotoxic and, in addition, failed to induce biologically significant increases in cellular proliferation in the liver, urinary bladder and thyroid gland on feeding to young adult Wistar rats. Nevertheless, it has been reported to enhance the yield of liver tumors when fed to rats or mice that developed an appreciable background incidence of these tumors without treatment. In order to resolve this situation, cell proliferation in response to BHT treatment was studied in enzyme-altered foci (EAF) induced in male Fischer 344 rats using the Solt-Farber procedure. It was demonstrated that feeding 0.5% dietary BHT for 30 days after the induction of EAF led to a 20- to 30-fold increase in the gamma-glutamyltranspeptidase-positive areas in both DEN- and saline-initiated rat livers, but to no major effects in glutathione S-transferase placental form (GSTP)-positive foci. Cell proliferation rates within EAF and surrounding normal liver were measured using different histological techniques. Nuclear labeling with [3H]thymidine and proliferating cell nuclear antigen (PCNA) over the total hepatocyte population indicated that BHT approximately doubled nuclear labeling in rats initiated with DEN. PCNA labeling in GSTP-positive foci was not affected by BHT. In GSTP-positive foci, evaluation of nucleolar organizer regions (AgNOR), which reflect cell proliferative in addition to transcriptional activity of ribosomal RNA, was achieved using a novel double staining technique. BHT diet did not affect the number of AgNOR per nucleus or the percentage AgNOR area/nucleus. Nevertheless, both PCNA labeling and the AgNOR area per nucleus were significantly greater in GSTP-positive foci compared with non-focal regions in rats fed either BHT or control diets. These results are discussed in the light of further experimental work required to determine the relevance of these data to possible human risk assessment for BHT.

Umbelliferous crop plants, including the parsnip (Pastinaca sativa L.), elaborate enhanced levels of furocoumarins, including psoralens, when subjected to biotic or abiotic stress. These furocoumarins are recognized to lead to phototoxicity. In this study, the effect of these agents, which are present in diseased parsnip root tissue, on the liver and two tissues on the route of entry to the body (the oesophagus and forestomach) were investigated. Young male Swiss Webster mice were fed for approximately 30 days with modified AIN-76A diets containing 32.5% dried healthy, 32.5% apparently healthy or 32.5% fungicide-treated parsnip root tissue, and 8, 16 or 32.5% dried diseased (Phoma complanata-infected) parsnip root tissue. As controls, three modified AIN-76A diets differing in their edible starch-to-sucrose ratios (C1-C3) were administered for an equal time. Dried healthy parsnip root tissue, compared with controls, did not significantly affect any of the indices of cellular proliferation or histopathological parameters that were assessed. Histopathological examination of the oesophagus and forestomach demonstrated no significant changes as a result of feeding any of the diets containing parsnip tissue. In the liver, the highest level (but neither of the two lower levels) of dried diseased parsnip root tissue led to swelling of the cytoplasm in cells surrounding the central vein of hepatic lobules, with consequent compression of the peripheral cells. Using [3H]thymidine radioautography, a dose-related increase in cell labelling with the level of diseased parsnip root tissue was demonstrated in the liver. Compared with control diet C2 only, the extent of [3H]thymidine labelling in the liver was increased in mice receiving apparently healthy parsnip tissue; a slight, not statistically significant, increase was also noted with fungicide-treated parsnip tissue. Increased [3H]thymidine labelling with the feeding of diseased parsnip tissue was also found in the greater curvature of the forestomach and the region of the oesophageal-forestomach junction, but not at the glandular junction of the forestomach nor in the mid-oesophagus.

Young virgin female Swiss Webster mice were fed AIN-76A semi-purified diets containing equal weights of different fats for approximately 30 days. Using [3H]thymidine radioautography, it was established that mice fed 100% lard or high levels of fish oils (menhaden oil or cod liver oil) developed elevated cellular proliferation in the duct cells of the mammary gland and an increased number of labeled cells/crypt in the crypts of the colo-rectum accompanied by an increase in the size of the proliferative compartment. A possible inverse correlation between the level of [3H]thymidine labeling in the mammary gland, but not in the colo-rectum, and the linoleic acid content of individual diets may help to explain the significance of these observations. The effect of adding an antioxidant mixture to these diets was to reduce the excess proliferation induced in the intestinal crypts by lard or fish oil to the level induced by soybean oil, but only partially so in the duct cells of the mammary gland.

Increased cellular proliferation has been associated with the enhanced expression of several key stages in carcinogenesis. A standard protocol was used to investigate the effect of specific dietary regimens on cellular proliferation. Young adult Swiss Webster mice were fed for 30 days with modified AIN-76A semi-purified diets designed to illustrate the effects of the levels of dietary or calorie restriction, different fibers and bulking agents, and different fats on cellular proliferation. Female mice were used for the restriction and fat studies, males for the fiber and bulking agent studies. Vaginal smears were taken from females from treatment day 15, and the mice killed 2 days following the first estrus following 30 days feeding; males were killed on the 30th day. One hour before death, mice were injected ip with 0.25 micro Ci/g 3[H]-thymidine. Slides were prepared for radioautography and histopathology. Both dietary and calorie restriction led to reduced 3[H]-thymidine labeling indices in each of the seven tissues studied, the mammary gland being the most severely affected. Different fibers and bulking agents, in specific cases, reduced labeling in the duodenum but not to a consistent statistically significant extent in the colon or colo-rectal region. In the duodenum, oat bran and oat gum were the most effective while wood cellulose (alphacel) had no effect. Investigations on the effects of different fats is continuing. High levels of lard, menhaden oil, or cod liver oil as the fat component of the AIN-76A diet, led to much higher levels of labeled cells in the mammary gland or colo-rectal region than did fat components rich in vegetable oils. The labeling indices appeared to be inversely correlated with the level of linoleic acid in the diet, a presumption that has been confirmed by investigating a series of diets containing different levels of this acid. Anti-oxidants were not used in any of these fat-modified diets. The overall results obtained in these studies clearly indicate the utility of cellular proliferation studies in investigating the effects of dietary modifications.

Forestomach neoplasia induced by the apparently non-genotoxic carcinogens, butylated hydroxyanisole and propionic acid, appears to arise by way of sustained high levels of cellular proliferation. Several other inducers of enhanced cellular proliferation, or the consequential incidence of hyperplastic lesions, have been identified in the rodent forestomach but the requisite carcinogenicity bioassays remain undone. In other tissues, such as the male rat kidney, the rodent thyroid follicular cell and the bladder epithelium, there is also evidence supporting the concept that sustained enhanced cellular proliferation may be an important early marker for non-genotoxic carcinogens. This reaction is, however, not likely to be the only marker necessary for the identification of non-genotoxic carcinogens.

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.

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.

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.

Male Wistar rats were dosed daily by gavage for 200 days with either (1) aspirin, 200 mg/kg; (2) acetaminophen, 200 mg/kg; (3) aspirin and acetaminophen, 200 mg/kg of each; (4) aspirin and acetaminophen, 100 mg/kg of each or (5) vehicle alone. None of the treatments produced any marked signs of toxicity and no drug related deaths were observed. The full dose combination (3) did significantly reduce the weight compared to the control group (5). When urinary lactic dehydrogenase and alkaline phosphatase were measured, all treatments caused some increase in activity but those containing aspirin had the greatest effect. Urinary pH and osmolarity were not effected by the treatments. Complete pathological examination of the animals failed to detect any significant changes when compared to the controls. It is concluded that there is no evidence of a toxic interaction between aspirin and acetaminophen in the rat.

The toxicological effects of analytical-grade hexachlorobenzene (HCB) were examined in two chronic studies. Study I was an in utero exposure carcinogenicity feeding experiment in which Sprague-Dawley rats, in groups of 40 males and 40 females except where noted, were fed from weaning on diets containing 0.0 (64 M/64 F), 0.32, 1.6, 8.0 or 40.0 (66 M/66 F) ppm HCB. After 3 months on test, the F0 rats were bred and 50 pups (F1) of each sex were randomly selected from every group. From weaning, when the F0 animals were killed, the F1 animals were fed their parents' diet for the rest of their life (130 wk). There were no treatment-related effects on growth, feed consumption, haematological parameters or survival in either generation. Increased heart and liver weights were found in the 8.0 and 40 ppm F0 males. HCB had no effect on fertility but pup viability was significantly reduced in the 40 ppm group. Histopathological changes in the F1 generation included significant linear trends in the incidence of parathyroid adenomas and phaeochromocytomas in both sexes, neoplastic liver nodules in females, centrilobular basophilic chromogenesis of the liver in both sexes, peliosis of the liver in females, peribiliary lymphocytosis of the liver in males and chronic nephrosis of the kidney in males. In Study II, the toxicological effects of HCB were examined as a consequence of varying the dietary levels of vitamin A. In this single generation lifetime (119 wk) feeding study, groups of 50 weanling Sprague-Dawley male rats were randomly assigned to each of the following dietary groups: control, control + 40 ppm HCB, 1/10 the vitamin A content of the control diet, 1/10 vitamin A + 40 ppm HCB, 10 times the vitamin A content of the control diet and 10 times vitamin A + 40 ppm HCB. After 25 and 49 wk on test, five animals from each group were killed and subjected to haematological and histological examinations. All other aspects of evaluation were similar to those for the F1 generation in Study I. No consistent differences were observed in the haematological parameters and there were no significant differences in the incidence of pathological lesions between the test groups. The animals in the 1/10 vitamin A groups, with or without HCB, had significantly lower body weights and poorer survival than did their corresponding control (normal vitamin A) groups.

Male Wistar rats were fed a purified basal diet with 20% lard and corn oil (3:1), sunflower oil, poppyseed oil, low-erucic rapeseed oil from Brassica napus, cultivar Tower, or mixtures of these oils, for 1 or 26 weeks. None of the hearts exhibited lipidosis at 1 week. At 26 weeks, the level of serum triglycerides was higher in rats fed the mixture of lard and corn oil than in those fed only vegetable oil, and the incidence of cardiac necrosis and fibrosis was higher in rats fed the rapeseed oil than in the other animals. Cardiac phospholipids in rats fed the rapeseed oil contained an elevated level of omega 3 docosahexaenoic acid, particularly in phosphatidylethanolamine. Poppyseed oil exhibited properties similar to those of sunflower oil, was absorbed as well as olive oil, and appeared to be a promising oil for human consumption.

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.

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.

Four infant monkeys were dosed orally with 500 microgram Hg/kg body wt./day /as methylmercury (MeHg) chloride dissolved sodium carbonate) beginning at 1 day of age. Neurological and behavioral signs of MeHg toxicity and blood Hg levels were monitored weekly. At first sign of MeHg intoxication, dosing with MeHg was terminated and the infants were monitored to assess reversal of the signs of MeHg toxicity. The first signs of MeHg toxicity, exhibited as a loss in dexterity and locomotor ability, were observed after 28--29 days of treatment; the blood Hg levels were 8.0--9.4 microgram Hg/g blood. Dosing was terminated at 28--29 days of treatment but the signs of MeHg toxicity continued to develop. The infants became ataxic, blind, comatose and were necropsied at 35--43 days after initiating treatment with MgHg. The mercury concentrations in tissues analyzed after necropsy were highest in liver (55.8 +/- 3.2 microgram Hg/g) followed by occipital cortex (35.6 +/- 4.8 microgram Hg/g) renal cortex (32.8 +/- 1.6 microgram Hg/g). The frontal and temporal cortices had 27.0 +/- 3.4 and 29.6 +/- 4.9 microgram Hg/g respectively while the cerebellar Hg concentration averaged 13.0 +/- 1.5 microgram Hg/g. The mean blood/brain ratio was 0.21 +/- 0.4. Histopathologic lesions were marked in the cerebrum with less severe lesions in the cerebellar nuclei. The Purkinje and granular cells of the cerebellar vermis appeared histologically normal. Lesions were not observed in the peripheral nervous system. The signs of MeHg intoxication, the tissue distribution of MeHg and histopathologic lesions observed in the infant monkeys were similar to those reported for adult monkeys.