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There is speculation that dietary polyphenols can provide cardioprotective effects due to direct antioxidant or antithrombotic mechanisms. We report in vitro and postingestion ex vivo effects of cocoa procyanidins, a procyanidin-rich cocoa beverage and dealcoholized red wine (DRW) on human platelet activation. In a series of in vitro studies, cocoa procyanidin trimers, pentamers or DRW (3 and 10 micromol/L) were incubated with citrated peripheral whole blood in the presence and absence of platelet agonists. Platelet activation was detected using fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (referred to herein as PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin). The percentage of CD42a-positive platelets coexpressing PAC-1 binding and/or CD62P was determined by multiparameter flow cytometry. Procyanidin trimers, pentamers and DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression. In contrast, procyanidin trimers, pentamers and DRW inhibited the platelet activation in response to epinephrine. The effects on platelet activation of cocoa beverage and DRW consumption were also studied in healthy subjects. Citrated blood was obtained before and 2 and 6 h after the ingestion of a cocoa beverage, a caffeine-containing beverage, DRW or water. Platelet activation was measured by flow cytometry. The consumption of DRW did not affect the expression of activation-dependent platelet antigens, either unstimulated or after ex vivo activation with epinephrine. However, the consumption of DRW increased PAC-1 binding in response to 100 micromol/L ADP ex vivo. Cocoa consumption reduced platelet response to agonists ex vivo. The ingestion of water had no effect on platelet activation, whereas a caffeine-containing beverage augmented the response of platelets to epinephrine. In summary, select cocoa procyanidins and DRW added to whole blood in vitro increased expression of platelet activation markers in unstimulated platelets but suppressed the platelet activation response to epinephrine. In contrast, cocoa consumption suppressed unstimulated and stimulated platelet activation in whole blood. This suppressive effect observed on platelet reactivity may explain in part the reported cardioprotective effects of dietary polyphenols.

Evidence from epidemiological studies suggests that a diet high in plant foods and rich in polyphenols is inversely associated with a risk for cardiovascular and other chronic diseases. Chocolate, like red wine and green tea, is a polyphenol-rich food, primarily containing procyanidin polyphenols. These polyphenols are hypothesized to provide cardioprotective effects due to their ability to scavenge free radicals and inhibit lipid oxidation. Herein, we demonstrate that 2 h after the ingestion of a procyanidin-rich chocolate containing 5.3 mg total procyanidin/g, of which 1.3 mg/g was (-)-epicatechin (epicatechin), plasma levels of epicatechin increased 133 +/- 27, 258 +/- 29 and 355 +/- 49 nmol/L in individuals who consumed 27, 53 and 80 g of chocolate, respectively. That the rise in plasma epicatechin levels was functionally significant is suggested by observations of trends for dose-response increases in the plasma antioxidant capacity and decreases in plasma lipid oxidation products. The above data support the theories that in healthy adults, 1) a positive relationship exists between procyanidin consumption and plasma procyanidin concentration and 2) the rise in plasma epicatechin contributes to the ability of plasma to scavenge free radicals and to inhibit lipid peroxidation.

We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase., (Copyright 2000 Academic Press.)

Background: Epidemiologic studies have shown inverse associations between dietary polyphenols and mortality from coronary heart disease. However, the basis for this protective association is uncertain. Food polyphenols reportedly have antioxidant properties and decrease platelet function in vitro., Objective: This study sought to evaluate whether consumption of a polyphenol-rich cocoa beverage modulates human platelet activation and primary hemostasis., Design: Peripheral blood was obtained from 30 healthy subjects before and 2 and 6 h after ingestion of a cocoa beverage (n = 10), a caffeine-containing control beverage (n = 10), or water (n = 10). Platelet activation was measured in terms of expression of activation-dependent platelet antigens and platelet microparticle formation by using fluorescent-labeled monoclonal antibodies and flow cytometry. Primary platelet-related hemostasis was measured with a platelet function analyzer., Results: Ex vivo epinephrine- or ADP-stimulated expression of the fibrinogen-binding conformation of glycoprotein IIb-IIIa was lower 2 and 6 h after consumption of cocoa than before consumption. Cocoa consumption also decreased ADP-stimulated P-selectin expression. In contrast, epinephrine-induced platelet glycoprotein IIb-IIIa expression increased after consumption of the caffeine-containing beverage but not after water consumption. Platelet microparticle formation decreased 2 and 6 h after cocoa consumption but increased after caffeine and water consumption. Primary hemostasis in response to epinephrine in vitro was inhibited 6 h after cocoa consumption. The caffeine-containing beverage inhibited ADP-induced primary hemostasis 2 and 6 h after consumption., Conclusions: Cocoa consumption suppressed ADP- or epinephrine-stimulated platelet activation and platelet microparticle formation. Cocoa consumption had an aspirin-like effect on primary hemostasis.

The morbidity and disability associated with autoimmune diseases represent a significant health problem. One in 31 people have one form or another of an autoimmune disease. Despite the avalanche of molecular data, immunogenetic definitions and improvements in serologic diagnosis, we are still far from discovering the etiologies of these diseases. For some autoimmune diseases, there may be a very long latency period between disease onset and clinical presentation. Existing therapies tend to be only partially successful and often accompanied by a variety of serious side effects. However, even in the absence of a complete understanding of the underlying genetic, environmental and coincidental factors that confer susceptibility to autoimmune diseases, we believe that it is possible to devise successful therapies by interfering with one or more of the pathways of destruction characteristic of a specific autoimmune disease. We have prepared a futuristic look at the treatment of autoimmune disease by extrapolation of current research directions as well as thoughts on new methods of delivery of recombinant monoclonal antibodies. We predict that we will "cure" autoimmune pathology long before we understand the etiology. In the case of inflammatory bowel disease, as a model, and taking advantage of what is known on animal studies, we illustrate the progress that has been made in elucidating these pathways of destruction and speculate about possible therapeutic approaches.

Caspase activity is a hallmark of apoptosis. Given that maternal zinc (Zn) deficiency results in apoptosis in the rat embryo, we assessed caspase activity in Zn-deficient embryos. Mid-gestation rat embryos were collected from dams fed either a Zn-deficient (0.5 Zn/g) diet ad libitum, or a Zn-adequate (25 microg Zn/g) diet ad libitum or pair fed to dams fed the Zn-deficient diet. Embryos from dams fed the Zn-adequate diet had a normal level of cell death, while embryos from the dams fed the Zn-deficient diet had either increased or normal levels of cell death. Zn-deficient embryos displaying increased cell death had increased caspase activity. Embryos with normal levels of cell death, regardless of maternal diet, had similar caspase activities. Thus, Zn-deficiency-induced apoptosis in vivo is associated with increased caspase activity., (Copyright 2000 Academic Press.)

It has been postulated that one mechanism underlying zinc deficiency-induced tissue alterations is excessive cellular oxidative damage. In the present study we investigated if zinc deficiency can induce oxidative stress in 3T3 cells and trigger select intracellular responses that have been associated to oxidative stress. Cells were exposed to control media or to chelated media containing 0.5, 5, or 50 microM zinc for 24 or 48 h. The oxidative status of the cells was evaluated as an increase in the fluorescence of the probe 5(or 6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate (DCDCDHF). After 24 and 48 h of exposure, the fluorescence intensity was significantly higher (4- to 15-fold) in the 0.5 and 5 microM Zn groups compared to the 50 microM Zn and control groups. The activity of the antioxidant enzymes CuZn (CuZnSOD) and Mn (MnSOD) superoxide dismutases was significantly higher in the 0.5 and 5 microM Zn cells compared to the 50 microM Zn and control groups at both the 24 and 48 h time points. These higher activities were associated with higher levels of MnSOD mRNA. After 24 h in culture, the level of activated AP-1 was markedly higher in the 0.5 and 5 microM Zn cells than in the control (72 and 58%, respectively) and 50 microM Zn cells (73 and 60%, respectively). NF-kappaB binding activity was lower in the 0.5 and 5 microM Zn cells than in controls. Thus, oxidative stress is induced by zinc deficiency in 3T3 cells. This oxidative stress results in an upregulation of oxidant defense mechanisms.

Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1 beta. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1 beta gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1 beta expression of PHA-stimulated cells by 1-15%, while the larger oligomers (pentamer-decamer) increased expression by 4-52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.

Primate species demonstrate a prolonged period of development before reproductive maturity that includes distinctive periods of rapid growth in the late fetal, late infancy and early adolescent stages. Rhesus monkeys resemble humans in this discontinuous pattern of growth and also in its relationship to brain development. Studies of zinc deprivation in rhesus monkeys have suggested an important relationship among growth rate, nutrient status and behavioral performance in infancy as well as adolescence. Recently, moderate combined zinc and iron deprivation (intake 0.2 mg Zn and 0.8 mg Fe/d, compared with control intake of 2.9 mg Zn and 1.7 mg Fe/d) during the adolescent growth spurt (29-32 mo. of age) of female rhesus monkeys (n = 8/group) was shown to influence behavior without affecting growth. Behavioral assessments included the Continuous Performance Test, the Delayed Nonmatch to Sample Test and activity (measured with an actimeter). The behavioral syndrome was characterized by reduced activity, reduced participation in behavioral testing and slower response. These changes could be reversed or prevented to some extent by altering the diet to include tablets of powdered beef (adding approximately 1.7 mg Zn and 0.7 mg Fe to daily intake). The study suggests that behavior may be sensitive to the quality of the diet available during the period of rapid adolescent growth and development.

This study investigates whether supplementation with topical RRR-alpha-tocopherol (Eol), topical RRR-alpha-tocopheryl succinate, and oral RRR-alpha-tocopheryl acetate can reduce the incidence of acute and chronic damage to the skin (i.e., sunburn and pigmentation and skin cancer, respectively) induced by ultraviolet (UV) irradiation to mice. Groups of twenty Skh:2 female hairless pigmented mice were treated with 1) lotion vehicle, 2) 5% Eol lotion, 3) 5% topical RRR-alpha-tocopheryl succinate lotion, or 4) lotion vehicle and oral RRR-alpha-tocopheryl acetate. Within each group, 15 mice were exposed to 0.24 J/cm2 of UV-B radiation three times per week. The animals' weights and food intakes were monitored, and the vitamin E concentrations of skin, liver, and adipose tissue were measured to determine whether the topical Eol resulted in significant tissue levels. Skin pigmentation was scored, and the total number of clinically detectable skin tumors per animal was counted weekly. Results showed that the skin concentrations of Eol, as well as levels in the adipose tissue, were increased after topical application. Mice treated with each form of vitamin E showed no signs of toxicity and had significantly less acute and chronic skin damage induced by UV irradiation, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancer.

Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

An excessive use of vitamin/mineral supplements is considered by many to be a common health problem. We surveyed 1,355 adolescent boys and girls attending athletic high schools in Korea for their usage patterns of vitamin/mineral supplements. The usage rate of the vitamin/mineral supplements was 35.8%. The most favored supplements were vitamin C, multivitamins, and calcium. The reasons most cited for taking supplements were "to recover from fatigue," and "to maintain health." Vitamin and mineral intakes occurred over a wide range; mean intake values were typically higher than the Korean RDA. Vitamins B1, B12 and C were consumed in very high amounts at 29.7, 17.9 and 11.1 times the Korean RDA, respectively. When the intakes of nutrients from supplements and diet were combined, it was observed that the intakes of niacin, folic acid, vitamin C, and iron exceeded levels that have been proposed as upper safe limits. The above data underscore the need to provide sound nutritional education to athletic adolescents and their coaches with respect to the use of vitamin/mineral supplements and the links between adequate diet, good health, and physical performance.

Transferrin (Tf) is known primarily for its role in the transport and cellular uptake of iron (Fe). Tf is also the major serum binding protein for Al. In this study, primary rat oligodendrocyte, neuron and astrocyte cultures were found to differ in Tf mediated Fe and Al uptake and in the effect of Al-Tf on Fe-Tf uptake during 4 h incubation periods. When incubated with Al-Tf (1.25 microM), oligodendrocytes displayed a 3- to 4-fold increase (p=.0002) in Al, neurons demonstrated a much smaller (p=.06) increase, and no increase was seen for astrocytes. When incubated with equimolar Al citrate or Al chloride, no increase in cellular Al was seen in any of the three cell types. Oligodendrocytes, astrocytes and neurons all demonstrated greater 59Fe uptake from Fe-Tf than Fe chloride. This uptake could be inhibited by excess Fe-Tf in oligodendrocytes and neurons, but not astrocytes. A small but significant inhibition of 59Fe uptake from Fe-Tf was seen after addition of Al-Tf to the incubation medium of oligodendrocytes, but not neurons or astrocytes. Oligodendrocytes may be particularly vulnerable to the accumulation of excess intracellular Al, and to interference of Al with Fe uptake. Such effects could contribute to Al-induced neurotoxicity if they result in altered myelin formation or maintenance.

Background: The adolescent growth spurt and menarche increase iron and zinc needs and could precipitate functional deficiencies if dietary sources are inadequate., Objective: The effects of mild, combined zinc and iron deprivation during the growth spurt and the ability of meat as a common dietary source of zinc and iron to reverse these effects was studied., Design: Pubertal female rhesus monkeys were fed control diets (n = 8) or diets marginally deficient in zinc (2 microg/g diet; n = 8) and iron (10 microg/g diet; n = 8) for 3 mo. A powdered beef supplement (104 microg Zn/g and 43 microg Fe/g, 11 +/- 2 g/d) was then fed daily to half of the deprived group for 3 additional months., Results: Growth and hematology were not affected significantly by iron-zinc deprivation, but plasma zinc and iron were somewhat lower in the deprived group than in the control group after 3 mo. The deprived monkeys reduced their participation in behavioral testing, responded more slowly and less frequently to test stimuli, and were less active. The beef supplement increased participation in testing and stabilized activity levels, but response times remained depressed. Plasma ferritin was lower in the nonsupplemented deprived monkeys than in the controls by the end of the experiment. Four of 8 of the deprived monkeys had iron deficiency anemia compared with none of the controls and 1 of 8 who received the beef supplement., Conclusions: Marginal zinc and iron deprivation in early adolescence can lead to behavioral and hematologic dysfunction in nonhuman primates and dietary beef supplements can prevent and reverse some of these effects.

Marginal iron deficiency is a common nutritional disorder in human populations world-wide; however, the neurobehavioral effects of chronic marginal iron (Fe) intakes during development are poorly characterized in animals. For this reason, we investigated whether mice reared on marginal Fe diets during pre- and postnatal development would experience neurobehavioral deficits. Swiss-Webster mice reared on either control (75 ppm Fe) or marginal iron (12.5 ppm) diets were assessed for changes in behavior on postnatal days 30, 40, and 50 using a neurobehavioral test battery. Because alterations in tissue mineral status can lead to an oxidative stress, markers of both protein (glutamine synthetase) and lipid oxidation (TBARS) were measured. Marginal iron animals exhibited a 20-55% reduction in grip strength. Although both marginal iron males and females demonstrated persistent lowering of body weights, statistical analysis using weight as the covariate demonstrated that the grip strength reductions were independent of body weight changes. This reduction in grip strength occurred in conjunction with a 25-45% lowering of brain iron in the marginal iron animals. Despite dramatic reductions in both brain and liver iron, hematocrits were unaffected by dietary iron reductions. Oxidative stress was indicated by an elevation in noniron-stimulated TBARS in the cerebellum of marginal iron animals. These data suggest that a chronic marginal Fe deficiency during critical periods of growth can result in functional changes in motor development even in the absence of iron deficiency anemia; furthermore, alterations in mineral status and oxidative stress may be mechanisms contributing to these observed changes.

The aims of this work were as follows: 1) to determine whether a purified diet currently used for studies with rats was acceptable for reproductive studies in frogs; and 2) to determine whether frogs are sensitive to a deficit of boron (B) in the diet. Adult Xenopus laevis were fed a nonpurified beef liver and lung (BLL) diet (310 microg B/kg), a purified diet supplemented with boron (+B; 1850 microg B/kg), or a purified diet low in boron (-B; 45 microg B/kg) for 120 d. Frogs fed the BLL and +B diets produced 11.3 and 12.2% necrotic eggs, respectively. Abnormal gastrulation occurred in <4% of the fertilized eggs in both groups, and 96-h larval survival exceeded 75% in both groups. In contrast, frogs fed the -B diet for 120 d produced a high proportion of necrotic eggs (54%). Fertilized embryos from the -B diet-fed frogs showed a high frequency of abnormal gastrulation (26.8%), and >80% of the embryos died before 96 h of development. Mean embryo cell counts at X. laevis developmental stage 7.5 (mid-blastula) were significantly lower in the -B embryos than in the BLL or +B embryos. BLL and -B embryos grown in low boron culture media had a high frequency of malformations compared with embryos grown in boron-supplemented media. These studies show that a purified diet that has been used in rodent studies was acceptable for reproduction studies in X. laevis. This work also demonstrates that a diet low in boron markedly impairs normal reproductive function in adult X. laevis, and that administration of the low boron diet results in an increase in both incidence and severity of adverse effects. In addition, these studies demonstrate the usefulness of the X. laevis model in nutrition studies.

The aim of this study was to determine the effect of dietary carbohydrate type on the expression of zinc (Zn) deficiency in rats with respect to tissue oxidative damage and defense mechanisms. Rats were fed diets containing adequate (+Zn) or low concentrations (-Zn) of Zn. Both fructose- and glucose-based diets were tested. Pair-fed controls were also studied to evaluate changes in the oxidative defense system which are secondary to Zn-deficiency-induced anorexia. Plasma and liver Zn concentrations and CuZn superoxide dismutase activities were lower in the -Zn rats than in the +Zn rats. Liver glutathione (GSH) and disulfide glutathione concentrations were higher in the -Zn rats than in the +Zn rats; this difference was most pronounced in the fructose groups. Liver and heart selenium glutathione peroxidase (Se-GSH-Px) activities were lower in the -Zn-fructose group than in the +Zn-fructose group. Liver Se-GSH-Px activity was higher in the fructose groups than in the glucose groups. Liver GSH reductase (GSH-Red) activity was lower in the -Zn-fructose group than in its control group. Liver glutamine synthetase activity was lower in the -Zn-glucose group and in the fructose groups than in the glucose control group. Liver thiobarbituric acid reactive substance (TBARS) production was similar among the groups. Collectively, these results support the concept that Zn deficiency can result in an impaired oxidant defense system. Based on the observation that pair-fed control animals also showed evidence of oxidative damage, we suggest that one factor that contributes to the effect of Zn deficiency is the reduction in caloric intake that occurs in these animals. Fructose feeding resulted in increased activities of several of the oxidant defense enzymes. Protein oxidative damage assessed by glutamine synthetase activity was increased by both Zn deficiency and fructose feeding.

We tested the hypothesis that zinc deficient animals would be characterized by an increased sensitivity to cadmium-induced oxidative damage to the testes. Weanling male rats were given free access to either a control (25 microg Zn/g) or a zinc deficient (0.5 microg Zn/g) diet; or restricted access to the 25 microg Zn/g diet at a level of intake similar to that of rats fed the 0.5 microg Zn/g diet. After 14 days on the diets, animals were injected s.c. with either saline or CdCl2 (2 mg Cd/kg body weight) solutions, and killed 24 h later. In the zinc-deficient group, testes weight and testes/body weight were higher in the cadmium-injected rats than in the saline-injected rats. The extent of hemorrhages, as indicated by high hemoglobin and testes iron concentrations was higher in the cadmium-treated zinc deficient group than in the cadmium-injected controls. In the zinc-deficient group, cadmium injection was associated with higher levels of lipid peroxidation (33% higher TBARS content) and protein oxidation (17% lower glutamine synthetase activity). Cadmium injection did not influence these parameters in the zinc-adequate groups. Extracellular superoxide dismutase activity was lower in the zinc-deficient group than in the zinc-sufficient groups; there was a trend (P < 0.06) for a lower activity in the cadmium- versus the saline-injected rats. These results support the concept that zinc deficiency increases the susceptibility of testes to cadmium-mediated free radical damage.

Puberty is a period of rapid growth and nervous system maturation, but it is little studied as a sensitive period for neurotoxicant effects. In this experiment, diets containing 7 (control), 100, 500, 750, or 1000-microg aluminum (Al.)/g diet as Al lactate and 3.2% citrate (to promote Al absorption) were fed to male mice beginning at puberty (45 days of age) for either 4 or 8 weeks. ANOVAs were conducted to identify group differences from control, and regression analysis with Al intake was used to evaluate dose-response trends. Dose-responsive effects of dietary Al on brain weight, Al, and Mn concentration, and on grip strength were seen at the end of the 4-week exposure. Although brain Al concentration was also elevated at the end of the 8-week exposure, no dose-responsive effects on other variables were noted. Neither exposure influenced auditory startle amplitude. The period after puberty was sensitive to dietary Al exposure, but affected variables apparently recovered when exposure continued into young adulthood.

Medicinal properties have been attributed to mushrooms for thousands of years. Mushroom extracts are widely sold as nutritional supplements and touted as beneficial for health. Yet, there has not been a critical review attempting to integrate their nutraceutical potential with basic science. Relatively few studies are available on the biologic effects of mushroom consumption, and those have been performed exclusively in murine models. In this paper, we review existing data on the mechanism of whole mushrooms and isolated mushroom compounds, in particular (1-->3)-beta-D-glucans, and the means by which they modulate the immune system and potentially exert tumor-inhibitory effects. We believe that the antitumor mechanisms of several species of whole mushrooms as well as of polysaccharides isolated from Lentinus edodes, Schizophyllum commune, Grifola frondosa, and Sclerotinia sclerotiorum are mediated largely by T cells and macrophages. Despite the structural and functional similarities of these glucans, they differ in their effectiveness against specific tumors and in their ability to elicit various cellular responses, particularly cytokine expression and production. Unfortunately, our data base on the involvement of these important mediators is still rather limited, as are studies concerning the molecular mechanisms of the interactions of glucans with their target cells. As long as it remains unclear what receptors are involved in, and what downstream events are triggered by, the binding of these glucans to their target cells, it will be difficult to make further progress in understanding not only their antitumor mechanisms but also their other biological activities.

There has been increased interest in the study of nutrition and immunity. This is especially true with respect to the hypothesis that consumption of specific foods may reduce an individual's susceptibility to the establishment and/or progression of immunologic disease. Although an increased intake of a specific food may improve health status in select cases, chronic consumption of large amounts of one specific food may in fact be detrimental. The studies described here examined the long-term effect of yogurt consumption on two different age populations, young adults (20-40 y) and senior adults (55-70 y). There were three study groups per age group, live-culture yogurt, pasteurized yogurt and control (no yogurt), given 200 g/d of yogurt for 1 y. The subjects completed a questionnaire detailing health parameters on a weekly basis and a 4-d food record was taken monthly. Blood was taken every 3 mo and complete blood chemistry, blood count, total and specific immunoglobulin (Ig)E, and interferon-gamma (IFN-gamma) production measured. Yogurt consumption, especially for the live-culture groups, was associated with a decrease in allergic symptoms in both age groups. Seniors in the control group experienced an increase in both total and LDL cholesterol, whereas those in the yogurt groups remained stable during the course of the study. There was little effect on IFN-gamma and IgE production, although seniors in the yogurt group had lower levels of total IgE throughout the year.

The present study focused on whether serum extracellular superoxide dimutase (EC-SOD) activity can be used as a functional indicator of marginal zinc deficiency in humans. Subjects in this study were 444 healthy adults over 30 yr of age living a normal rural life in Kyunggi province, Korea. The mean dietary zinc intake of subjects obtained from one 24-h recall was 6.41 +/- 4.35 mg and the average serum zinc concentration of the subjects was 11.06 +/- 2.44 micromol/L. Subjects were divided into three groups by serum zinc concentrations: adequate (serum zinc >10.7 micromol/L), low (serum zinc 9.0-10.7 micromol/L), and very low (serum zinc <9.0 micromol/L) groups. A total of 50 subjects were selected from the three groups for analysis of EC-SOD activities. The EC-SOD activity of subjects increased with increasing serum zinc concentrations, and the activities of the three groups were significantly different as indicated by the Kruskal-Wallis test (p = 0.0239). Also, serum EC-SOD activities were significantly correlated with serum zinc concentrations (r = 0.289, p = 0.04). Serum EC-SOD activities, however, were not significantly correlated to the dietary zinc intakes. In conclusion, these results show that EC-SOD activities are decreased in subjects with low serum zinc concentrations and suggest that EC-SOD activity may be a functional indicator of zinc nutritional status in humans.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.

In experimental animals, dietary manganese deficiency can result in numerous biochemical and structural abnormalities. Deficient animals can be characterized by impaired insulin production, alterations in lipoprotein metabolism, an impaired oxidant defense system, and perturbations in growth factor metabolism. If the deficiency occurs during early development, there can be pronounced skeletal abnormalities and an irreversible ataxia. Several lines of evidence suggest that manganese deficiency may be a problem in some human populations. Manganese toxicity can also pose a significant health risk. In experimental animals, acute manganese toxicity can result in numerous biochemical pathologies. However, the above occurs typically when the manganese is given via injection; most animals show considerable resistance to dietary manganese toxicosis. Similarly, confirmed cases of manganese toxicity in humans are currently restricted to cases of exposure to high levels of airborne manganese, and to cases when manganese excretory pathways are compromised.

The present study further investigates evidence for lipid peroxidation in atherosclerotic aortic tissue by determining the activity of antioxidant enzymes and concentrations of lipid peroxide fluorochromes in abdominal aortas from 15 patients with abdominal aortic aneurysms (AAA), an additional 7 patients with ruptured abdominal aneurysms, and 12 patients with atherosclerotic occlusive disease (AOD). Aortas from nonatherosclerotic organ donors served as nondiseased controls. Cu, Zn-superoxide dismutase (Cu,Zn-SOD) activities in AAA and AOD tissues were 16% and 25% of control activity, respectively. Mn-SOD activity in diseased aortae were about 65% of controls. CuZn-SOD protein in AAA and AOD was 56% and 100% of controls, respectively, resulting in significantly lower CuZn-SOD specific activity in these tissues. Ruptured AAA tissue also had low SOD activity and protein. Glutathione peroxidase (GPx) activity in AAA and AOD aortas was 70% and 65% of controls, respectively, and glutathione reductase (GR) activity in AAA and AOD aortas was 80% and 65% of control activities, respectively. These results were associated with significantly higher lipid peroxide fluorochromes, expressed as U/g aorta, in both groups of atherosclerotic aortas than in controls. AOD aortas had 33% higher fluorescence than AAA aortas, but the highest levels were seen in ruptured AAA. These data further support the involvement of free radicals and lipid peroxidation in atherosclerotic aortic disease, but do not indicate that these mechanisms are specifically involved in aneurysm formation versus development of occlusive disease.

To date, boron (B) essentiality has not been conclusively shown in mammals. This article summarizes the results of a series of in vitro and in vivo experiments designed to investigate the role of B in mammalian reproduction. In the first study, rat dams were fed either a low (0.04 microg B/g) or an adequate (2.00 microg B/g) B diet for 6 wk before breeding and through pregnancy; reproductive outcome was monitored on gestation day 20. Although low dietary B significantly lowered maternal blood, liver, and bone B concentrations, it had no marked effects on fetal growth or development. The goal of the second study was to assess the effects of B on the in vitro development of rat postimplantation embryos. Day 10 embryos collected from dams fed either the low or adequate B diets for at least 12 wk were cultured in serum collected from male rats exposed to one of the two dietary B treatments. Dams fed the low B diet had a significantly reduced number of implantation sites compared to dams fed the B-adequate diet. However, embryonic growth in vitro was not affected by B treatment. The aim of study 3 was to define the limits of boric acid (BA) toxicity on mouse preimplantation development in vitro. Two-cell mouse embryos were cultured in media containing graded levels of BA (from 6 to 10,000 microM). Impaired embryonic differentiation and proliferation were observed only when embryos were exposed to high levels of BA (>2000 microM), reflecting a very low level of toxicity of BA on early mouse embryonic development. Study 4 tested the effects of low (0.04 microg B/g) and adequate (2.00 microg B/g) dietary B on the in vitro development of mouse preimplantation embryos. Two-cell embryos obtained from the dams were cultured in vitro for 72 h. Maternal exposure to the low B diet for 10, 12, and 16 wk was associated with a reduction in blastocyst formation, a reduction in blastocyst cell number, and an increased number of degenerates. Collectively, these studies support the concept that B deficiency impairs early embryonic development in rodents.

Based on reports that copper deficiency can result in altered arachidonic acid (AA) metabolism and the knowledge that AA can modulate insulin secretion, we investigated the interaction of copper deficiency and AA on glucose-stimulated insulin secretion by using isolated islets. Lean male (Fa/Fa) Zucker rats were fed either a copper-adequate (Cont) or low-Cu (Cu Defic) diet (8.7 vs. 0.5 micrograms copper/g diet) for 5 weeks. Pancreatic islets were isolated and incubated in medium containing 3.3, 8.3, or 16.7 mM glucose with or without 100 microM AA. Insulin secretion was measured after either 5 (phase I) or 45 (phase II) min of incubation. In the absence of AA, islets from Cu Defic rats secreted more insulin during phase I than those from Cont rats; phase II insulin secretion was similar between the groups. Exogenous AA treatment increased insulin secretion from islets during both phases of insulin secretion, regardless of dietary copper treatment. In the presence of AA, islets from Cu Defic rats secreted more insulin in phase I than did islets from Cont rats at the 3.3 and 8.3 mM glucose levels. Similar findings were made for phase II insulin secretion. Consistent with this, Cu Defic rats tended to have higher plasma insulin levels than Cont rats (472.6 vs. 199.6 pM). In summary, Cu deficiency increases insulin release in isolated islets; this response is particularly pronounced when AA is present in the incubation medium.

Young male rats subjected to a dietary manganese (Mn) deficiency respond to the deficiency by reducing their growth rate. The growth hormone (GH)/insulin-like growth factor (IGF) axis is critical for linear growth; this system is exquisitely sensitive to the nutritional state of the animal. In this study, we examined circulating GH, IGF-1, and insulin levels in Mn-deficient (-Mn; fed a 0.5 microg Mn/g diet) and sufficient (+Mn; fed a 45 microg Mn/g diet) male Sprague-Dawley rats. Additionally, we examined the distribution of circulating IGF binding proteins (IGFBPs) in animals of both dietary groups as these proteins modulate IGF-1 action in vivo and in vitro, and have been demonstrated to be altered in a number of nutritional and physiological states. Body weight was significantly reduced in -Mn relative to +Mn rats. Consistent with other studies, daily food intake was not altered. However, cumulative food intake (over 3 months) was marginally lower in -Mn versus +Mn animals. -Mn animals displayed lower circulating concentrations of IGF-1 (66% of control levels) and insulin (60% of control levels) despite having significant elevations in circulating GH levels relative to +Mn animals (140% of control levels). The IGFBP profile of -Mn animals reflected their elevated GH status, as we observed increased binding of tracer (125I-IGF-1) to the circulating IGFBP-3 complex (120% of control binding) using native chromatography techniques. Interestingly, the lower circulating insulin concentrations of -Mn animals did not result in dramatic elevations in lower-molecular-weight binding proteins. In summary, we demonstrate that in young male rats, Mn deficiency is associated with alterations in IGF metabolism. These alterations may contribute to the growth and bone abnormalities observed in -Mn animals.

In the first part of the present study we investigated the effects of pre-natal and early postnatal exposure of mice to high levels of dietary Al3+ on myelin lipid composition and lipid oxidation. We found: (1) a significantly higher (104%; P<0.01) content of brain myelin galactolipids in the high-Al3+ group than in controls, and, (2) a significant correlation (r2=0.70; P<0.01) between the concentration of myelin galactolipids and TBARS (2-thiobarbituric acid-reactive substances) content, a parameter of lipid oxidation. Based on these results, we evaluated in an in vitro model (liposomes) whether galactolipids could affect the capacity of Al3+ to stimulate Fe2+-initiated lipid oxidation, and whether this effect could be due to the promotion of changes in membrane physical properties (membrane phase separation and rigidification). The presence of galactolipids (10-40 mol%) in the liposomes caused a concentration-dependent increase in the stimulatory effect of Al3+ on Fe2+-induced TBARS production, and on the ability of Al3+ to induce phase separation and membrane rigidification. The capacity of Al3+ (10-100 microM) to induce lateral phase separation in liposomes composed of phosphatidylcholine/phosphatidylserine/galactolipid (36:24:40, molar ratio) was correlated significantly (r2=0.99; P<0. 001) with the stimulatory action of Al3+ on Fe2+-induced TBARS production. We propose that the high content of galactolipids found in myelin from Al3+-intoxicated mice could favour Al3+-induced changes in membrane physical properties, with the subsequent acceleration of lipid oxidation rates.

Rat embryos (gestation days 9.0 and 10.0) obtained from dams that were fed a Cu-adequate (8 micrograms Cu/g) or Cu-deficient (< 0.5 micrograms Cu/g diet were cultured for 48 hr in Cu-adequate (16.2 microM) or Cu-deficient (1.0 microM) rat serum. Control embryos cultured in control serum were morphologically normal. Embryos from Cu-deficient dams developed abnormally when cultured in Cu-deficient serum; the abnormalities included distended hindbrains, blisters, blood pooling, and cardiac defects. Control embryos cultured in Cu-deficient serum and Cu-deficient embryos cultured in control serum also showed abnormal development, but to a lesser degree than that of the Cu-deficient embryos cultured in Cu-deficient serum. To test the idea that the above abnormalities were due in part to free radical induced damage occurring secondary to an impaired oxidant defense system, a chemiluminescence assay was used to detect superoxide dismutase (SOD) activity in the cultured embryos. SOD activity was lowest in embryos cultured in Cu-deficient serum. When the Cu-deficient serum was supplemented with antioxidants (CuZnSOD or glutathione peroxidase), its teratogenicity was reduced. These data support the idea that an impaired oxidant defense system contributes to the dysmorphology associated with Cu deficiency. However, the Cu-deficient embryos also had low cytochrome c oxidase activity compared to control embryos--thus, multiple factors are likely contributing to Cu deficiency-induced abnormalities.

It has been hypothesized that the developmental toxicity of certain compounds is, in part, due to maternal toxicity resulting in alterations in zinc (Zn) metabolism that affects the developing conceptus. In the present work the effects of developmentally toxic doses of 2-ethylhexanoic acid (EHXA), 2-ethylhexanol (EHXO), and valproic acid (VPA) on Zn metabolism were investigated in the pregnant rat. In experiment 1, dams were intubated with EHXA (3.13, 6.25, 9.38 or 12.5 mmol/kg), EHXO (6.25, 9.38 or 12.5 mmol/kg), VPA (1.56, 3.13, 6.25 or 9.38 mmol/kg), or corn oil (control; 1.0 ml/kg) at 14:00 h on gestation day (GD) 11.5, intubated with 32 microCi 65Zn at 22:00 h, and then killed at 08:00 h on GD 12.5. At the higher dose levels of EHXA and EHXO, and at all dosages of VPA, the percentage of 65Zn retained in maternal liver was higher, while that in the embryos was lower, than in controls. Chemical-associated changes in 65Zn distribution were associated with increased maternal liver metallothionein (MT) concentrations. In experiment 2, dams were fed diets containing 1, 25 or 97 microg Zn/g from GD 0-16 and intubated with 3.5 mmol EHXA or 1.0 ml corn oil/kg/d from GD 8-15. Dams were killed on GD 16 or 19. High incidences of encephalocele and tail defects were noted in the GD 16 fetuses of EHXA-treated dams fed either the low or adequate Zn diet, the highest incidences being in the low Zn group. On GD 19 the incidence of tail defects tended to be higher in the EHXA groups than in oil-treated controls, the highest incidence occurring in the low Zn EHXA group. Encephalocele was only observed in the low Zn EHXA-treated group. Fetal weight and crown-rump lengths were decreased by EHXA treatment and low dietary Zn. The incidence of rib anomalies was higher in the EHXA-exposed groups than in their respective oil controls. In experiment 3, GD 10.5 embryos collected from control dams were cultured for 48 h in serum from control or EHXA-treated male rats fed 4.5 or 25.0 microg Zn/g diets. Embryos cultured in either EHXA or low Zn sera exhibited delayed development; the addition of Zn to these sera eliminated their developmental toxicity. These results support the hypothesis that certain chemicals which induce maternal toxicity act, in part, to influence embryonic Zn metabolism and trigger abnormal development. Importantly, the teratogenic effects of these chemicals can be modulated by dietary Zn intake.

Trace element status is known to be altered in the diabetic state, although the factors affecting trace element homeostasis in this condition are not well understood. The authors examined the effects of a high fructose diet (40% wt:wt) vs a control diet on the copper (Cu), zinc (Zn), and iron (Fe) concentrations in the kidney, plasma, and red blood cells of islet transplanted (TX) and sham-operated (SHAM) rats. Male, Wistar Furth rats made diabetic by streptozotocin injection (55 mg/kg, iv) were given an intraportal islet transplant (1000 islets); control animals were sham-injected, sham-operated (SHAM). Rats within TX and SHAM groups were assigned to either a high fructose diet (40% fructose, 25% cornstarch, FR) or a purified control diet (33% cornstarch, 33% dextrose, CNTL) containing identical amounts of mineral mixture for a period of 6 wk. Kidney Cu concentration was significantly elevated among hyperglycemic TX-CNTL rats (224+/-25 nmol/g wet wt), but was markedly reduced in hyperglycemic TX-FR rats (109+/-14 nmol/g) relative to normoglycemic controls. This occurred in spite of similar levels of glucose, insulin (fed and fasted), insulin secretory capacity, body weight, and food intake in the TX-CNTL and TX-FR groups. Among the subgroup of rats with normal glucose levels post-TX, kidney Cu levels normalized and were unaffected by dietary treatment (normoglycemic TX-CNTL = 60+/-5 nmol/g; normoglycemic TX-FR = 40+/-2 nmol/g). Kidney Cu concentrations also were unaffected by fructose feeding in SHAM animals (CNTL, 60+/-4 nmol/g and FR, 51+/-5 nmol/g). Kidney Zn and Fe concentrations were similar among the treatment groups. Plasma and red blood cell (RBC) Cu, Zn, and Fe concentrations were also similar among the groups. Since fructose feeding led to a substantial reduction of kidney Cu concentrations in the presence of hyperglycemia, the authors suggest that this model can be useful in examining effects of altered kidney Cu accumulation in the diabetic animal.

A review of the literature provides support for the concept that maternal nutritional status has a significant influence on embryonic and fetal development. The consumption of `poor' diets has been shown to be a risk factor for poor pregnancy outcome, while the provision of selected nutritional supplements prior to and during pregnancy has been associated with improved pregnancy outcome. Despite the above, it has been difficult to identify specific nutrient deficiencies as causative factors of abnormal development. One explanation for this failure is that embryo/fetal nutritional deficiencies can arise through a number of mechanisms in addition to a low maternal intake of a nutrient(s). Evidence is presented for the hypothesis that the developmental toxicity of a number of teratogens can be ascribed, in part, to their ability to induce alterations in the partitioning of essential trace elements between the maternal and fetal unit. An implication of the above hypothesis is that maternal diet can be an important modulator of the developmental toxicity of several agents.

Di(2-ethylhexyl) phthalate (DEHP) is a peroxisome proliferator whose administration to rodents induces a pleiotropic response mediated by the peroxisome proliferator-activated receptor-alpha (PPAR alpha). The mechanisms underlying DEHP-induced reproductive toxicity and teratogenicity are not well understood but could be the result of an alteration in gene expression by PPAR alpha. Additionally, phthalate exposure is known to impair fetal zinc (Zn) levels during pregnancy. In this work, we investigated whether the reproductive toxicity and teratogenicity of DEHP are mediated by PPAR alpha and whether the receptor influences maternal and/or embryonic Zn metabolism. Pregnant female mice, homozygous wild-type (+/+) or PPAR alpha -null (-/-), were intubated with either vehicle alone or 1,000 mg DEHP/kg body weight on gestation day (GD) 8 and 9. Pregnancy outcome was evaluated on GD10 and GD18 in two cohorts of animals. Compared to controls, DEHP administration resulted in maternal toxicity, embryo/ fetal toxicity, and teratogenicity in both (+/+) and (-/-) mice. Maternal liver mRNA for cytochrome P-450 4A1 (CYP4A1) was higher in DEHP-treated (+/+) mice but not in DEHP-treated (-/-) mice on GD10, consistent with their respective phenotype. Maternal liver MT and Zn levels were significantly higher than in controls on GD10. In addition, embryonic Zn content was significantly lower in both genotypes treated with DEHP compared to controls. Results from this work show that DEHP-induced reproductive toxicity, teratogenicity, and altered Zn metabolism are not mediated through PPAR alpha-dependent mechanisms. In addition, this work suggests that DEHP-induced alterations in Zn metabolism contribute to the mechanisms underlying DEHP-induced reproductive toxicity and teratogenicity.

The administration of alpha-hederin, an inducer of metallothionein, results in a secondary zinc deficiency that may be an important maternally mediated mechanism of developmental toxicity. Previous studies have shown adverse developmental outcome with a single administration of alpha-hederin to rats on gestation day (GD) 8 or 11. The objective of this study was to determine whether dosing of alpha-hederin throughout organogenesis would result in a sustained elevation of maternal hepatic metallothionein and subsequent developmental abnormalities. Rats were administered dosage levels of 0 (vehicle only), 20, or 30 mumol/kg from GD 6-15. Maternal hepatic metallothionein levels were 10-fold higher on GD 16 in the treatment groups than the controls. Consequently, liver zinc concentrations increased 60% and 54%, whereas plasma levels decreased 23% and 33% in the 20 and 30 mumol/kg treatment groups, respectively. At GD 20, mean fetal weights of the treatment litters were 11% less than control litters. The administration of alpha-hederin resulted in a threefold increase in the number of offspring that exhibited developmental abnormalities, including visceral and skeletal malformations. Following an oral pulse of 65Zn subsequent to treatment with 0 or 20 mumol/kg of alpha-hederin, the distribution of 65Zn to the liver of treated dams was twice that of controls, whereas the radiolabeled zinc apportioned to the decidua and uterus decreased by 44%. Furthermore, the 65Zn detected in the embryos from treated dams was 70% lower than in embryos from control dams. In conclusion, low doses of a metallothionein inducer administered to the dam from GD 6-15 resulted in a sustained elevation of hepatic metallothionein and a subsequent redistribution of zinc leading to a decrease in the zinc available to the embryo and ultimately to adverse development of the offspring. Repeated dosing throughout organogenesis, as required in regulated safety assessment testing, increased the severity of the effects previously observed with single large dosages of the toxicant administered during midgestation.

Leuprolide acetate in depot form (0.75 mg/kg body weight/month, i.m.) was administered to four female rhesus monkeys from 18-30 months of age, a period that includes the premenarchial growth spurt. They were compared to eight age matched controls. As anticipated, sexual maturation was blocked in the Leuprolide group and menarche did not occur. Growth was also severely retarded; no weight gain occurred during the study in the Leuprolide group as compared to a 25% weight gain (P = .044) in the control group. The Leuprolide group also lost muscle mass. Food intake normalized for body weight was not affected. Linear growth averaged 35% less in the Leuprolide group. Serum IGF-1 concentrations increased from 486 +/- 84 to 965 +/- 47 ng/mL (P = .0025) in the Leuprolide group and from 838 +/- 139 to 3,006 +/- 545 ng/mL (P = .0016) in the control group. These data suggest that premenarchial pituitary/gonadal suppression results in a distinctive pattern of growth retardation in monkeys.

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation.

The capacity of Al3+ to promote oxidative damage to brain membranes was investigated both in vitro and in vivo. In vitro, Al3+ and related metals (Sc3+, Ga3+, In3+, Be2+, Y3+, and La3+) stimulated Fe2+-initiated lipid and protein oxidation in brain myelin and synaptic membranes. Al3+, Sc3+, Y3+, and La3+ significantly promoted protein-associated carbonyl production in myelin, while in synaptic membranes, the stimulatory effect was observed in the presence of Ga3+, In3+, Y3+, Sc3+, and La3+. In myelin the magnitude of the stimulation of lipid oxidation followed the order Sc3+, Y3+, La3+ > Al3+, Ga3+, In3+ > Be2+. When compared to mitochondria and microsomal and synaptic membranes, myelin showed a marked susceptibility to Al3+-mediated lipid peroxidation. The differential susceptibility of myelin compared to synaptic membranes could not be explained by differences in membrane composition, since the relative content of negatively charged phospholipids (binding sites) was similar for both membranes, and myelin had a lower content of poly-unsaturated fatty acids (substrates of lipid oxidation) and a higher concentration of alpha-tocopherol compared to synaptic membranes. In a model of Al3+ intoxication imposed to mice during pregnancy and early development, a 72% higher content of lipid peroxidation products was found in brain myelin. The fluidity of myelin evaluated by the polarization fluorescence of 1,3-diphenylhexatriene was significantly higher in the Al3+-intoxicated mice than in controls. Since myelin has a high relative content of lipid:protein compared to other membranes, these results support our hypothesis that ions without redox capacity can stimulate in vitro and in vivo lipid oxidation by promoting phase separation and membrane rigidification, thus accelerating lipid oxidation.

Objective: To establish similarities or differences in tissue concentrations of zinc, copper, and iron in Bull Terriers with lethal acrodermatitis (LAD) and tail-chasing behavior (TCB) and to confirm the suspicion that copper is involved in the etiopathogenesis of LAD., Samples: Serum samples from 29 Bull Terriers (9 control dogs, 6 dogs with LAD, 14 dogs with TCB), and liver and kidney specimens from 2 dogs and 1 and 4 dogs with LAD or TCB, respectively., Procedure: Serum, liver, and kidney mineral (zinc, copper, and iron) concentrations in Bull Terriers with LAD or TCB and in a group of control dogs were analyzed, using flame atomic absorption after wet ashing technique., Results: Serum zinc and copper concentrations were lower (P < 0.05) in dogs with LAD, compared with values for control dogs and dogs with TCB. Liver zinc and copper concentrations were similar to serum values. Kidney zinc and copper concentrations were similar among the 3 groups. Serum, liver, and kidney iron concentrations had a wide range of variability within all 3 groups., Conclusion: Copper deficiency is associated with LAD. The primary cause of LAD may be copper deficiency, with zinc involved secondarily, or combined zinc and copper deficiencies. The role of ion deficiency in TCB was not clarified., Clinical Relevance: Serum zinc and copper concentrations should be determined when LAD is suspected.

To investigate the influence of coffee consumption on iron, zinc and copper status, 64 Sprague-Dawley female rats were assigned to four treatment groups: nonpregnant-given coffee, nonpregnant-given water, pregnant-given coffee and pregnant-given water. The coffee groups received a freeze-dried coffee solution (1.1% w/v) as the sole source of liquids during the 18-day study period; all groups were provided a purified diet marginal in iron ad libitum. On day 18 (of gestation in the pregnant rats), rats were intubated with 0.033 MBq59Fe after an 8 h fast and killed 4, 8, 12 or 24 h post-gavage. Fluid intake was significantly lower in the coffee groups. Coffee did not affect food consumption, weight gain, hemoglobin or hematocrit in either pregnant or nonpregnant rats. In nonpregnant rats, the percentage of 59Fe was lower in plasma and higher in the spleen and erythrocytes in the coffee group than in controls; in pregnant rats the coffee group had a higher percentage of 59Fe in the stomach (at 4 h only), blood, spleen and bone than controls. There were no significant effects of coffee on tissue concentrations of Fe, Zn or Cu in the nonpregnant group, but pregnant rats given coffee had high concentrations of Fe in the liver, bone and placenta, and low placenta Zn concentrations compared to controls. In fetuses, there were trends towards lower weight, length and percentage 59Fe, but higher liver Fe, in the coffee group than in the controls. The results suggest that coffee intake leads to (a) increased attempts at erythropoeisis, an indication of iron deficiency, in both nonpregnant and pregnant rats, and (b) impaired placental Fe transport.

It is well recognized that deficiencies of essential trace elements during early development can result in structural abnormalities and/or embryonic death. Recently, there has been increasing interest in the concept that small excesses of essential metals can also have negative effects on the developing embryo. We hypothesized that, with respect to toxicity, metals with similar physico-chemical properties would act by similar mechanisms to influence the preimplantation embryo. In the current study we investigated the influence of four essential (Cu, Mn, Fe, Zn), and eight nonessential (Cr, Hg, Pb, V, Al, Ag, Cd, As) metals on mouse preimplantation embryonic development. Two cell stage mouse embryos were cultured for 72 h in media containing varying metal concentrations (0.05 - 200 microM). Embryo cell differentiation and proliferation were respectively assessed by scoring for blastocyst formation and final embryo cell number. Both nonessential and essential metals were embryotoxic at relatively low concentrations. However, in contrast to our expectations, at similar molar concentrations, redox active essential metals were less toxic than non-redox active nonessential metals. These data suggest that direct metal binding to critical membrane sites and/or intracellular ligands, including protein and nucleic acids, may trigger abnormal development and death prior to metal-associated oxidative damage.

It is well established that in mammals transitory zinc (Zn) deficiency during embryogenesis can have a negative influence on fetal development. Similar to Zn deficiency, maternal exposure to low levels of ionizing radiation during the first day of pregnancy has been shown to negatively affect preimplantation embryo development, and higher doses of maternal irradiation during late stages of embryogenesis can result in malformations. Here we report the effect of transitory maternal Zn deprivation combined with low dose irradiation during embryogenesis on fetal outcome. Pregnant mice were acutely dosed with 0.00, 0.05, 0.10, or 0.25 Gy of gamma-radiation the day after mating (GD0), or with 0.00 or 0.50 Gy on GD8. Mice irradiated on GD0 were either fed a low Zn diet (0.4 microgram Zn/g) for 48 h prior to mating through GD0 or GD5 and then switched to a control diet (50 micrograms Zn/g) through GD18, or they were fed the control diet throughout gestation. Dams irradiated on GD8 were either fed the low Zn diet from GD0 through GD10 and then switched to the control diet, or they were fed the control diet throughout gestation. Zn deprivation did not influence any of the maternal or embryonic/fetal parameters measured in the cohorts fed the low Zn diet through GD0. In contrast, groups fed the low Zn diet through GD5 or GD10 had lower mean maternal body weights, fewer live fetuses/litter, and a higher incidence of resorptions than controls. Fetuses from dams fed the low Zn diet through GD10 were smaller and shorter, and had a higher frequency of malformations than controls. Irradiation on GD8 under adequate Zn conditions had no effect on any parameter, but GD8 irradiation during maternal Zn deficiency had an additive effect on the frequency of fetal malformations.

Al is found in the developing conceptus, but little information is available concerning its tissue distribution and its changes in concentration with age. Because Al has affinity for many of the same biological ligands as the essential mineral cations Ca, Mg, Zn, Fe, and Mn, we hypothesized that Al might show a pattern of developmental concentrations that was similar to one or more of these elements in the brain, a major target of Al toxicity. Concentrations of Al, Ca, Mg, Zn, Fe, and Mn were measured in spinal cord, brainstem, cerebellum, and forebrain of guinea pig fetuses on gestation day (GD) 30 and 45, at birth, and on postnatal day (PND) 3, 6, and 12. Dams were fed commercial guinea pig chow, which contained 47 micrograms Al/g. Tissue Al and Mn were measured with electrothermal atomic absorption spectrophotometry (ETAAS), and the other elements with inductively coupled axial plasma spectroscopy (ICAP-AES). Al concentrations in the brain regions were highest in spinal cord, brainstem, and cerebellum, and decreased during late gestation and lactation. Al did not show marked increases in regional brain concentrations during the final third of gestation as did Fe, Mg, and Zn. In contrast to Fe and Ca, Al did not accumulate in placenta. Al was the only element to show higher concentrations in spinal cord than in any other tissue at birth. In summary, the tissue distribution of Al did not follow that of essential cations as examined in this study.

The proposed increased use of methanol (MeOH)-based fuels raises the concern for an increased risk for MeOH toxicity. MeOH, which is detoxified in part via a folate-dependent pathway, is known to be teratogenic in rodents. Previous observations have implicated maternal folate status as a critical modulator for the developmental toxicity of MeOH. The current study extends these findings, examining the effect of maternal dietary folate intake on fetal folate stores, as well as identifying a possible marker for the prediction of the developmental toxicity of MeOH. Virgin female CD-1 mice were assigned to diets containing either 400 (marginal) or 1200 (control) nmol folic acid (FA)/kg, and and 1% succinylsulfathiazole for 5 weeks prior to mating and throughout breeding and gestation. From gestation day (GD) 6 through 10 dams were given by gavage deionized, distilled water (dH2O) or MeOH at 2.5 g/kg body weight, twice daily. On GD 18, mice were weighed and killed and the liver, kidneys, and gravid uteri removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted; fetuses were weighed individually and examined for cleft palate and exencephaly. The marginal FA dietary treatment resulted in low maternal liver (50% reduction) and red cell folate (30% reduction) concentrations, as well as low fetal tissue folate concentrations (60 to 70% reduction) relative to the adequate FA dietary groups. Marginal FA treatment alone resulted in cleft palate in 13% of the litters; there were no litters affected with cleft palate in the adequate FA-control group. Marginal FA-MeOH treatment resulted in a further increase in the litters affected by cleft palate (72% of litters affected). The percent of litters affected by exencephaly was highest in the marginal FA-MeOH group. The frequency of micronuclei in maternal and fetal reticulocytes, a marker for chromosomal abnormalities, was not influenced by either the marginal FA diet or by MeOH treatment. These results show that marginal folate deficiency in pregnant dams significantly increases the teratogenicity of MeOH.

Methanol, which is detoxified via a folic acid-dependent pathway, has been shown to be teratogenic in mice. Given recent observations that the level of dietary folic acid intake may be inversely related to the occurrence of select birth defects in humans, we tested the hypothesis that dietary folic acid intake would influence the developmental toxicity of methanol. Virgin female mice were fed one of three diets containing 400 (low), 600 (marginal), or 1,200 (adequate) nmol folic acid/kg diet for 5 weeks prior to and following mating. On gestation days (GD) 6-15, dams were administered by gavage either vehicle (distilled, deionized water) or methanol at 2.0 or 2.5 g/kg body weight, twice daily. On GD 18, mice were weighed and killed and the liver, kidneys, and gravid uteri removed and weighed. Implantation sites, live and dead fetuses, and resorptions were counted; fetuses were weighed individually and examined for cleft palate and exencephaly. One third of the fetuses in each litter were examined for skeletal morphology. Maternal liver folate concentrations were approximately 40-50% lower in the low dietary folic acid groups than in the marginal and adequate groups; methanol did not affect maternal liver folate concentration at term. Maternal net gestational weight gain was lowest at the lowest dietary folate level but was not affected by methanol. Gravid uterus weights were lowest in the low dietary folic acid groups exposed to the high methanol dose and the number of live fetuses per litter was lowest in the low folic acid groups. Fetal body weights were lowest in the low folic acid groups and significantly lower in the methanol groups relative to vehicle-treated animals. Fetal crown-rump lengths were shorter in the methanol-treated groups; this parameter was not affected by folic acid treatment. Both methanol and low dietary folic acid increased the incidence of cleft palate, with the highest number of affected litters in the low dietary folic acid group. These results support the concept that maternal folate status can modulate the developmental toxicity of methanol.

Previous studies have demonstrated that zinc deficiency can be associated with high rates of oxidative damage to testes lipids, proteins, and DNA in male rats. In the present work, different aspects of the oxidant defense system (enzymes and lipid-soluble antioxidant substances) were characterized in the testes of control and zinc-deficient rats. Seventeen-day-old males were given free access to either a control (25 microg Zn/g) or a zinc-deficient (0.5 microg Zn/g) diet, or the 25 microg Zn/g diet at a level of food intake similar to that of zinc-deficient rats. Animals were sacrificed 14 days after the initiation of the diet. The activities of copper-zinc superoxide dismutase (CuZn SOD) and glutathione reductase (GRed) were significantly higher (34% and 23%, respectively) in testes from the zinc-deficient animals than in those of the ad libitum controls. In testes, the activities of manganese superoxide dismutase (Mn SOD) and glutathione peroxidase (GPx), and the concentration of alpha-tocopherol and ubiquinol-9 and -10 were similar among the groups. However, the ratio of reduced/total concentration of both ubiquinols was higher in the zinc-deficient and restrict-fed animals than in the ad libitum controls. Testes homogenates from the zinc-deficient rats showed a low susceptibility to Fe(II)-induced oxidation, which could be explained in part by a lower peroxidation index, mainly due to the decreased testicular content of the fatty acid 20:4 observed in these animals. In summary, both undernutrition and zinc-deficiency can cause an oxidative stress situation in testes, for which cells tend to compensate by increasing select components of the oxidant defense system.