The granular layer of Tomes in experimental caries in rats.

In examination of ground sections of human third maxillary molar teeth, the granular layer of Tomes was shown to consist of expansion of dentinal tubules. The microradiographic density was decreased well inside the layer. It was postulated that the findings were expressions of dentin resorption. The theory was tested experimentally in rats fed a cariogenic diet with low calcium and phosphorus. The morphology of mandibular molar teeth was studied by electron probe microanalysis and by microradiography. It was concluded that the primary event in cariogenesis was an expansion of peripheral dentinal tubules with formation of confluent microcavities and, thus, an unmasking of the granular layer of Tomes, which normally is only potential. A subsequent loss of mineral density in the outer enamel eventually caused breakdown of the outer enamel with caries visible from the surface. The loss of density within the enamel was postulated to result from interruption of the normal flux of nutrients, metabolites and ions between the dentin and enamel. Whereas an increase in dietary calcium and phosphorus delayed and reduced significantly, but did not prevent cariogenesis, it supported the theory that experimental caries in rats is a metabolic disease with the peripheral dentin the primary target.

Effects of different dietary carbohydrates on hepatic enzymes of copper-deficient rats.

The present study was undertaken to measure the activities of several hepatic enzymes of regulatory importance in the pathways of lipogenesis and gluconeogenesis in rats fed diets marginally deficient in copper (1.2 micrograms Cu/g of diet) and containing either fructose, glucose, or starch as the carbohydrate sources. Although all copper-deficient rats exhibited the characteristic signs of copper deficiency, they were more pronounced in rats fed the diet containing fructose. Except for the activity of phosphoenolpyruvate carboxykinase which was unaffected either by copper deficiency or by the type of dietary carbohydrate, the hepatic activities of glucose-6-phosphate dehydrogenase, malic enzyme, L-alpha-glycerophosphate dehydrogenase and fructose 1,6-diphosphatase were unaffected by copper deficiency but were affected by the type of carbohydrate in the diet. Fructose produced the greatest increase in enzymatic activities, whereas starch produced the least activity and glucose induced an intermediate effect. These results indicate that the deleterious effects of a fructose diet deficient in copper on biochemical and physiological indices could not be due to an immediate metabolite of fructose. However, the involvement of a subsequent metabolite of fructose in the mechanism of copper utilization and/or requirement cannot be excluded.

The severity of copper deficiency in rats is determined by the type of dietary carbohydrate.

The purpose of this investigation was to study the interaction between copper and dietary carbohydrates on clinical and enzymatic indices associated with copper deficiency. Copper deficiency was produced in rats by feeding diets adequate in all nutrients including selenium and chromium, but marginal in copper (1.2 micrograms/g diet) containing 62% of either starch, fructose, or glucose. During the fifth week, the fructose of the copper-deficient diet (20 rats) was replaced by either starch (10 rats) or by glucose (10 rats). The experiment was terminated after 11 weeks. Copper deficiency in rats fed fructose significantly lowered body weight and hematocrit, but increased liver weight, blood urea nitrogen, ammonia, cholesterol, and triglycerides when compared to rats fed starch or glucose. The copper metalloenzyme, superoxide dismutase, the selenoenzyme, glutathione peroxidase, and hepatic ATP were decreased in the copper-deficient rats fed fructose as compared to copper-deficient rats fed starch or glucose. These results indicate that fructose may be the dietary component which has a deleterious effect on copper and selenium status. Changing the type of dietary carbohydrate in copper-deficient rats from fructose to either starch or glucose ameliorated the severity of the deficiency. The protective effects were more pronounced with starch than with glucose.

Effect of dietary carbohydrates and copper status on blood pressure of rats.

Copper deficiency was induced in rats by feeding diets containing either 62% starch, fructose or glucose deficient in copper for 6 weeks. All copper deficient rats, regardless of the dietary carbohydrate, exhibited decreased ceruloplasmin activity and decreased serum copper concentrations. Rats fed the fructose diet exhibited a more severe copper deficiency as compared to rats fed either starch or glucose. The increased severity of the deficiency was characterized by reduced body weight, serum copper concentration and hematocrit. In all rats fed the copper adequate diets, blood pressure was unaffected by the type of dietary carbohydrate. Significantly reduced systolic blood pressure was evident only in rats fed the fructose diet deficient in copper. When comparing the three carbohydrate diets, the physiological and biochemical lesions induced by copper deprivation could be magnified by feeding fructose.

The interaction of type of dietary carbohydrates with copper deficiency.

The present study was designed to determine if the more severe copper deficiency in rats fed sucrose and fructose, as compared to starch, is due to a specific effect of the fructose or to a nonspecific effect of any simple carbohydrate. Seventy weanling male rats were fed, for 9 wk, copper-deficient diets or copper-supplemented diets containing either 62% starch, fructose, or glucose. Decreased hematocrit, serum copper, and ceruloplasmin concentrations but increased heart and liver weights, total liver lipid, and hepatic iron concentrations were found in all copper-deficient rats regardless of the dietary carbohydrate. Feeding rats the high glucose diet decreased plasma albumin and liver glycogen but increased blood urea nitrogen when compared to rats fed starch. However, rats fed fructose generally exhibited a more severe copper deficiency as compared to rats fed either starch or glucose. The severity was characterized by lower (p less than 0.05) body weight, liver glycogen, hematocrit, serum copper, and albumin. Conversely, liver and heart weights, blood urea nitrogen, and plasma glutamic oxaloacetic transaminase were higher (p less than 0.05). Plasma cholesterol was increased by copper deficiency only in rats fed fructose or glucose. During the study, 17 of the 40 rats fed copper-deficient diets died; 66% of those fed fructose, 26% fed glucose, and 30% fed starch. These results suggest that the fructose moiety of sucrose is responsible for the increased severity of copper deficiency in rats fed sucrose as compared to starch.

Impairment of glucose tolerance in copper-deficient rats: dependency on the type of dietary carbohydrate.

Copper deficiency was induced in weanling rats in order to study the possible interaction between the types of dietary carbohydrate and copper deficiency on glucose tolerance. Weanling male rats were fed copper-deficient or copper-supplemented diets containing either 62% starch, fructose or glucose. During week 5 the fructose portion of the copper-deficient diet was replaced (20 rats) by starch (10 rats) or glucose (10 rats). During the 9th week, an oral glucose tolerance test weas performed. Copper deficiency was associated with impaired glucose tolerance characterized by increased blood glucose and decreased insulin levels only in copper-deficient rats fed the monosaccharides fructose or glucose but not the polysaccharide starch. Changing the dietary carbohydrates in the copper-deficient diet from fructose to starch increased insulin levels and decreased blood glucose in response to the glycemic stress when compared to rats continuously fed fructose. Although both glucose and fructose feeding impaired the glucose tolerance, fructose was more diabetogenic. This could be demonstrated by some improvements in glucose tolerance when the copper-deficient rats were switched from the fructose to the glucose diet. The data indicate that copper deficiency per se does not impair glucose tolerance.

Interaction between dietary carbohydrate and copper nutriture on lipid peroxidation in rat tissues.

The effects of the interactions between dietary carbohydrates and copper deficiency on superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and their roles in peroxidative pathways were investigated. Weanling rats were fed diets deficient in copper and containing either 62% starch, fructose, or glucose. Decreased activity of SOD was noted in all rats fed the copper-deficient diets regardless of the nature of dietary carbohydrate. However, the decreased activity was more pronouced in rats fed fructose. Feeding the fructose diets decreased the activity of GSH-Px by 25 and 50% in the copper-supplemented and copper-deficient rats, respectively, compared to enzyme activities in rats fed similar diets containing either starch or glucose. The decreased SOD and GSH-Px activities in rats fed the fructose diet deficient in copper were associated with increased tissue per-oxidation and decreased hepatic adenosine triphosphate (ATP). When the fructose in the diet of copper-deficient rats was replaced with either starch or glucose, tissue SOD and GSH-Px activities were increased and these increases in enzyme activity were associated with a tendency toward reduced mitochondrial peroxidation when compared to the corre-sponding values for rats fed fructose throughout the experiment Dietary fructose aggrevated the symptoms associated with copper deficiency, but starch or glucose ameliorated them. The protective effects were more pronounced with starch than with glucose.

Role of dietary fructose in the enhancement of mortality and biochemical changes associated with copper deficiency in rats.

Rats were fed copper deficient (0.9 microgram/g) or copper-supplemented diets in which the carbohydrate was either starch, sucrose, or fructose (62% by weight) for 7 wk. Regardless of the nature of the carbohydrate, copper deficiency decreased blood ceruloplasmin activity, hepatic copper and ATP levels, and increased plasma cholesterol and triglycerides. Copper deficiency in rats fed sucrose or fructose, but not those fed starch, significantly lowered blood hematocrit, Hb, and albumin and significantly increased heart and liver weight and the glucose response to a glycemic stress. Hepatic copper level was significantly lower in copper-deficient rats fed sucrose or fructose than in those fed starch. Fasting blood glucose, cholesterol, and triglyceride levels were significantly higher in copper deficient rats fed fructose than in those fed starch. During the study 14 copper-deficient rats died, one of 10 fed starch, six of 20 fed sucrose, and seven of 20 fed fructose. Death was apparently the result of rupture of the heart in the region of the apex. These results indicate that fructose-containing carbohydrates as compared to starch markedly increase the severity of copper deficiency in rats. Whether this effect is due to differences in the nature of the simple carbohydrate (fructose versus glucose) or to molecular size (simple versus complex carbohydrate) remains to be established.

Effect of copper deficiency on metabolism and mortality in rats fed sucrose or starch diets.

Copper deficiency was induced in rats by feeding sucrose or starch diets deficient in copper. Copper-deprived rats fed either diet exhibited decreased plasma ceruloplasmin concentration and increased plasma cholesterol. Glucose homeostasis and utilization was impaired both in vivo and in vitro. Oral glucose tolerance was impaired, insulin binding decreased, and CO2 formation and lipogenesis from [U-14C]glucose were decreased. Feeding sucrose but not starch diets deficient in copper magnified the copper deficiency and resulted in 60% mortality. Although both deficient diets contained the same concentration of copper, the hepatic copper concentration of rats fed sucrose was reduced nearly threefold compared to rats fed starch. Reduced epididymal fat pad, increased liver weight, reduced blood hemoglobin and a marked hypertrophy of the heart with gross deformities as well as histopathologic changes were noted only in those rats fed the copper-deficient sucrose diet. The biochemical lesions induced by deprivation of copper can be suppressed by feeding diets containing starch or can be magnified by high sucrose intake.

Osmotic and peroxidative fragilities of erythrocytes from vitamin E-deficient lead-poisoned rats.

Weanling male rats were fed either a vitamin E-deficient Torula yeast diet fortified with selenium or the same diet supplemented with 100 ppm vitamin E. One group of rats fed each diet received plain distilled water, whereas another group received 250 ppm lead as lead acetate in the drinking water. After a 3 month feeding period, erythrocyte osmotic and peroxidative fragilities were determined in an osmotic test recorder. Dietary vitamin E had little or no effect on the osmotic fragility of red cells. Lead in the drinking water, however, decreased the osmotic fragility of red cells from deficient rats. Lead poisoning also markedly decreased the elevated peroxidative fragility characteristic of erythrocytes from vitamin E-deficient rats. This effect of lead in reducing the peroxidative fragility of red cells from deficient rats could be seen at levels as low as 25 ppm lead in the drinking water. Lead added in vitro decreased the peroxidative fragility of red cells from vitamin E-deficient non-poisoned rats, whereas neither mercury nor cadmium had such an effect. Lead may decrease the osmotic and peroxidative fragility of erythrocytes from vitamin E-deficient rats by "tanning" the red cell membrane. These results suggest that the peroxidative fagility test as carried out with an osmotic test recorder may not be a valid indicator of the vitamin E status of animals exposed to lead.

Comparative effects of selenium and vitamin E in lead-poisoned rats.

Weanling male rats were fed a Torula yeast diet supplemented with selenium, vitamin E, or both for 3 months. Of rats fed each diet, one group received 250 ppm lead in the drinking water and another group did not. In rats not poisoned with lead, neither vitamin E nor selenium deficiency affected spleen weight, hematocrit value, or erythrocyte mechanical fragility. Vitamin E deficiency increased the splenomegaly, anemia, and mechanical fragility of red cells of lead-poisoned rats, whereas selenium deficiency did not. Addition of 0.5 ppm selenium to the vitamin E-supplemented diet increased slightly the splenomegaly and anemia in lead-poisoned rats. Excess levels of selenium (2.5 and 5 ppm) in the vitamin E-deficient diet had little or no effect on spleen size or hematocrit of rats not receiving lead, but partially prevented the splenomegaly and anemia of red cells from either non-poisoned or lead-oisoned vitamin E-deficient rats, but not as effectively as vitamin E. These results show that vitamin E status of rats is more important that selenium status in determining response to toxic levels of lead. Excess dietary selenium did protect partially against lead poisoning in vitamin E-deficient rats, but the levels of selenium used were toxic in themselves.

Filterability of erythrocytes from vitamin E-deficient lead-poisoned rats.

The time required for red blood cells (RBC) from vitamin E-deficient lead-poisoned (-E + Pb) rats to pass through polycarbonate filters after incubation in vitro was much greater than that of RBC from vitamin E-supplemented non-poisoned rats. Vitamin E deficiency per se (i.e., in non-poisoned rats) often increased filtration times, but in all such experiments the RBC from -E + Pb groups had even longer filtration times. Administration of lead to rats supplemented with vitamin E had little effect on the filtration rate of RBC. N,N'-diphenyl-p-phenylenediamine (DPPD) prevented the increased filtration times characteristic of RBC from -E + Pb rats, but replacement of the lard in the vitamin E-deficient basal diet by more highly polyunsaturated fats did not exacerbate the increased filtration times of RBC from -E + Pb rats. The increased filtration time of RBC from -E + Pb rats appeared to be related to the extent of RBC lipid peroxidation. Decreasing the pH of the RBC incubation medium from 7.4 to 6.6, an acidity typical of the spleen, markedly increased the filtration times of RBC from -E + Pb rats. Addition of lead in vitro increased filtration times of RBC from both vitamin E-deficient and supplemented non-poisoned rats, but filtration times tended to be longer in the deficient group. These results suggest that vitamin E deficiency and lead toxicity act synergistically to alter the deformability of the RBC thereby rendering it vulnerable to sequestration in the spleen.

Lead poisoning in vitamin E-deficient rats.

Weanling male rats were fed either a vitamin E-deficient Torula yeast diet or the same diet supplemented with 100 ppm vitamin E for a period of 3 months. One group of animals fed each diet received 250 ppm lead in the drinking water, whereas another group of animals fed each diet received no lead in the water. Vitamin E deficiency per se had little or no effect on hematocrit value, reticulocyte count, spleen weight, or erythrocyte mechanical fragility in rats not poisoned with lead. On the other hand, the decreased hematocrit, increased reticulocyte count, and splenic enlargement due to lead poisoning were much more pronounced in vitamin E-deficient rats than in rats supplemented with vitamin E. The resistance to mechanical trauma of red blood cells from vitamin E-deficient lead-poisoned rats was much less than that of red cells from vitamin E-adequate lead-poisoned rats. Dietary vitamin E status had no significant influence on the increased mechanical fragility of erythrocytes from nonpoisoned rats caused by exposure to lead in vitro. These results suggest that vitamin E deficiency enhances the susceptibility of animals to the in vivo hemolytic effect of lead poisoning.

Fungi that infect cottonseeds before harvest.

As a part of an investigation of aflatoxins and other mycotoxins in cottonseeds at harvest, samples of seeds collected from the 1971 crop at locations across the U.S. Cotton Belt were examined to determine the kinds of microorganisms causing internal or seed-coat infection in the field. Aspergillus flavus infection was absent from all seeds examined from most areas but was present in some samples from Arizona, California, and Texas. Fusarium spp., Alternaria sp., and A. niger caused internal infection at many locations; Colletotrichum gossypii and Rhizopus stolonifer were present in seeds from some areas but were generally much less common. Many of the infections with A. niger were in the seed coat. Bacterial infections were fairly frequent. In a series of commerical samples from Arizona. A. flavus infection was found in 61% of seeds, with fiber showing the bright, greenish-yellow (BGY) fluorescence that is diagnostic for A. flavus boll rot. Aflatoxin contamination was also concentration in the same seeds. The above findings agree with previous data showing that aflatoxin contamination of cottonseeds before harvest occurs rarely, if at all, in most parts of the U.S. Cotton Belt and that when such contamination does occur, it tends to be concentrated in seeds with the BGY fluorescence in their fiber and seed fuzz.