Cadmium absorption in women fed processed edible sunflower kernels labeled with a stable isotope of cadmium, (113)Cd.

The apparent fractional absorption of cadmium (Cd) from sunflower kernels (SFK) was determined in women volunteers by using kernels labeled with a stable isotope of Cd ((113)Cd) by injecting it into the flowering head. Fourteen women who were between the ages of 30 and 70 years, who did not use tobacco products, who were in good health, and who had been consuming a self-selected diet low in Cd content participated in the study. The volunteers were fed a breakfast composed primarily of cereal, milk, and fruit juice. The breakfast also contained a portion of (113)Cd-labeled SFK processed into a buttery spread. Each volunteer collected individual stool samples for 21 days beginning immediately after they had consumed the labeled kernels. The total amounts of Cd and (113)Cd excreted in each stool were determined by isotope dilution inductively coupled plasma mass spectrometry. Mean fecal Cd excretion was 14.1+/-4.1 microg/day and mean (113)Cd absorption was 10.6+/-4.4%. In agreement with previous studies, no significant (P>0.3) correlation between Cd absorption and serum ferritin concentrations was found in women whose serum ferritin concentrations were >25 ng/mL. These data suggest that the availability of Cd from highly processed sunflower kernels to humans is similar to that reported for other types of food.

Cadmium bioavailability from edible sunflower kernels: a long-term study with men and women volunteers.

Sunflower kernels (SFK) contain natural Cd in somewhat higher amounts than most other foods. This study was designed to determine if a change in the body burden of Cd could be measured in volunteers who consumed a controlled amount of SFK for 48 weeks. Healthy men and women between 23 and 59 years of age were divided into three groups each by age, sex, and weight. For 48 weeks, one group consumed 255 g (9 oz) of SFK/week, a second group consumed 113 g SFK and 142 g peanuts/week, and a third group consumed 255 g peanuts/week. SFK contained 0.52 microg Cd/g and peanuts contained 0.11 microg Cd/g. The consumption of 255 g SFK/week increased the average estimated intake of Cd from approximately 65 to approximately 175 microg/week. Although the consumption of Cd-containing SFK significantly increased the estimated Cd intake, there were no significant changes in Cd concentration in red blood cells, Cd excretion in the urine, or Cd concentrations in new hair growth. However, fecal Cd excretion significantly increased as the amount of SFK consumption increased. N-Acetyl-beta-D-glucosaminidase, a marker for kidney dysfunctions, was not significantly changed by consuming SFK-Cd. Overall, the results suggest that the consumption of Cd in the form of SFK at 9 oz (255 g)/week for 48 week had no adverse effect on the body burden of Cd.

Differentiation of human U937 promonocytic cells is impaired by moderate copper deficiency.

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 microM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus interferon-gamma (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the host's defense system.

Idiorrhythmic zinc dose-rate induction of intestinal metallothionein in rats depends upon their nutritional zinc status(dagger).

The idiorrhythmic dose-rate feeding experimental model was used to study the induction of intestinal metallothionein (iMT) by zinc (Zn) in the gastrointestinal (GIT) mucosa of young growing male rats relative to their nutritional Zn status. The idiorrhythmic approach requires that the average dietary Zn concentration, referred to as modulo (M), is kept constant across different groups over the whole experimental epoch (E). This is done by adjusting the Zn concentration of the supplemented diet to compensate for the reduction in the number of days on which this diet is fed, the latter being spread evenly over the whole experiment. Idiorrhythms (I) involve offering the diet with n times the overall Zn concentration (M) only every nth day with a Zn-deficient diet offered on other days. We studied three modulos (low-Zn, M3; adequate-Zn, M12; and high-Zn, M48), each M having 8 analogous idiorrhythms (I = Mx/1 to 8Mx/8); every I was fed over a 48-d idiorrhythmic E. Over the wide range of peak doses of dietary Zn (3-384 mg Zn/kg diet), the higher the modulo, the greater the capacity for iMT to be induced (M3 < M12 < M48; P < 0.05). Also, the ability of Zn to induce iMT increased proportionally with the progression of the idiorrhythms from I = Mx/1 to 8Mx/8 (P < 0.001). When rats were fed M3, less Zn was required to induce iMT than when they were fed M12 or M48. Thus, within the M and E limits of this study, the better the nutritional Zn status of the animal, the more Zn is required to induce iMT and vice versa. The fact that iMT was increased means that the amount of available Zn was not proportional with the actual steady state of its metabolism. This indicates that for any Zn supplementation program to be effective, it should progress gradually from a lower to a higher Zn dose relative to the given nutritional Zn status.

Mineral status of female rats affects the absorption and organ distribution of dietary cadmium derived from edible sunflower kernels (Helianthus annuus L.).

The intake of food cadmium (Cd) in microg/day over time can increase the body burden of this element. Some human populations that consume subsistence rice-based diets low in calcium (Ca), iron (Fe), and zinc (Zn) are more susceptible to Cd poisoning than populations that consume more nutritious diets. This study determined the effects of marginal deficiencies of these essential elements on the absorption and organ retention of Cd from a natural food that contains Cd, edible sunflower kernels (Helianthus annuus L.; SFK). Weanling female rats were fed diets containing 20% SFK in a 2x2x2 factorial design with marginal and adequate amounts of Ca, Zn, and Fe. Marginal Zn (11 mg/kg) and Fe (13 mg/kg), and Cd (0.18 mg/kg) were derived solely from 20% SFK. These amounts of Fe and Zn represented 39 and 90% of the NRC requirement for the rat, respectively. The marginal dietary Ca concentration (2.5 g/kg) was one-half the NRC requirement. After 5 weeks on the experiment, rats were fed 1 g of their respective diets containing SFK extrinsically labeled with 37 kBq 109Cd, and absorption was determined by whole-body counting techniques. Rats were then killed and organs collected for 109Cd assays. No effect of treatment on weight gain was observed; however, when dietary Zn was low, feeding marginal Ca elevated Cd absorption by 50% (P<0.05) over those fed adequate Ca and Zn. Feeding marginal Fe elevated Cd absorption >2.5-fold (P<0.001) over those fed adequate Fe. In contrast, the naturally occurring Zn in SFK that provided 90% of the rat's requirement was enough to deter excessive absorption of Cd and enough to alone prevent significant elevation of organ Cd. Organ content of 109Cd and Cd followed the same general pattern as whole-body absorption. These data show that marginal nutritional deficiencies of Ca and Fe can readily enhance the body burden of Cd that comes from the diet. Also, some natural competitors of Cd, such as Zn, contained in foods can independently minimize Cd absorption.

Zinc-related metallothionein metabolism in bovine pulmonary artery endothelial cells.

Bovine pulmonary artery endothelial cells (BPAEC) were cultured in vitro under a variety of conditions to investigate how metallothionein (MT) might participate in zinc homeostasis. Experimental conditions included 10% serum to ensure that the in vitro environment would be a better reflection of the in vivo situation than with protein-free medium. MT was increased by acutely high zinc concentrations (100-200 micromol/L) in the extracellular environment. MT was relatively insensitive to moderate changes in zinc concentration (2-50 micromol/L), even after prolonged exposure for 7 to 12 days. BPAEC had reduced MT content when grown in medium containing serum that had been dialyzed to remove components with a molecular mass of less than 1,000, including zinc. Because the principal source of the major minerals in the experimental medium was not the serum, their concentrations in the final medium were not significantly influenced by serum dialysis. Restoring the zinc concentration in the medium containing the dialyzed serum did not restore MT content in BPAEC, suggesting that some small molecular weight molecule other than zinc established their basal MT content. This study did not identify these putative factors in serum, but hormones are likely candidates. Forty-eight-hour incubations of BPAEC with interleukin (IL-6) or dexamethasone increased cellular MT; however, 17beta-estradiol decreased MT, and IL-1 and adenosine 3',5'-cyclic phosphate (cAMP) had no discernible effect. We conclude that extracellular zinc concentrations have relatively little impact on the cellular concentrations of MT and zinc of BPAEC in vitro. Zinc homeostasis by BPAEC is not maintained by changing the MT concentration in response to changes in the extracellular zinc environment. (J. Nutr. Biochem. 10:00-00, 1999).

Physiologic concentrations of zinc affect the kinetics of copper uptake and transport in the human intestinal cell model, Caco-2.

Copper uptake was enhanced and copper transport was reduced in Caco-2 cells cultured in media containing high concentrations of zinc. Here we show that physiologic zinc concentrations also affect copper movement into and out of Caco-2 cells. Cells were seeded onto Falcon membranes with high pore density and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, nonessential amino acids, glucose and glutamine. In one experiment, the cells were exposed to media containing either 5 or 25 micromol zinc/L from d 14 to 21 after seeding. Then, copper uptake and transport, in both apical and basolateral directions, were measured by using 64Cu. Cells exposed to 25 micromol zinc/L had a 25% higher (P < 0.05) uptake of 64Cu from the apical side than those exposed to 5 micromol zinc/L. There was no effect of zinc on 64Cu uptake from the basolateral side, even though the amount of label taken up was as much as threefold higher (P > 0.05) than from the apical side. Transport of 64Cu across the cell layer in both directions was 50% less (P < 0.05) in cells exposed to 25 micromol zinc/L vs. 5 micromol zinc/L. In another experiment, zinc-exposed cells were labeled with 64Cu and efflux of the label to the apical and basolateral sides was measured over time. The rate of efflux to the apical side was linear and not affected by zinc. However, there was a 37% reduction (P < 0.05) in 64Cu efflux to the basolateral side by the higher zinc concentration. Curve-fit analysis showed that the basolateral efflux was made up of an exponential and a linear component. Cellular zinc concentrations were proportional to the zinc concentrations in the media. Although the data suggest that high media zinc inhibited the copper efflux transporter and enhanced the influx transporter, copper did not accumulate in the cell.

Cadmium burden of men and women who report regular consumption of confectionery sunflower kernels containing a natural abundance of cadmium.

Because of inherent genetic and physiological characteristics, the natural concentration of cadmium in the kernels of sunflowers grown in uncontaminated soils of the northern Great Plains region of the United States is higher than in most other grains. We tested the hypothesis that a habitual consumption of sunflower kernels will increase the body burden and health effects of cadmium in humans. Sixty-six men and women who reported consuming various amounts of sunflower kernels were recruited and divided by sex and kernel consumption: those who consumed less than or equal to 1 ounce(oz)/week and those who consumed more than 1 oz/week. Cadmium intake was assessed by calculation from 7-day food diaries, cadmium burden by whole blood cadmium, red blood cell (RBC) cadmium and urine cadmium concentrations, and health effects by urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) activity and beta2-microglobulin (beta2MG). The results showed that high intakes of sunflower kernels (>1 oz/day) significantly increased the intake of cadmium (p<0.004). However, the amount of cadmium in whole blood or RBCs was not affected by cadmium intake. Urinary excretion of cadmium also was not affected by cadmium intake. Urine NAG activity and the amount of urinary beta2MG were significantly elevated in the urine of high sunflower kernel consumers when the values were expressed on a urine volume basis (p<0.03), but not when expressed on a creatinine basis (p>0.05). Because normal ranges for the excretion of these protein markers have not been established, it was not possible to determine if these elevated values were meaningful. However, given the knowledge that habitual consumption of sunflower kernels with natural cadmium concentrations higher than most other food products will increase the average intake of dietary cadmium, the potential exists for an increased body burden of cadmium. Controlled feeding studies in humans should be pursued in order to determine if the body burden does indeed increase and, if so, is it a cause for concern.

Idiorrhythmic dose-rate variability in dietary zinc intake generates a different response pattern of zinc metabolism than conventional dose-response feeding.

We compared the effects of idiorrhythmic dose-rate feeding and conventional dose-response on the induction of intestinal metallothionein (iMT), expression of aortal heat-shock protein mRNA (HSP70mRNA) induced by restraint stress, and accumulation of Zn in the femur and incisor of young growing male rats. An idiorrhythmic approach requires that the average dietary Zn concentration (modulo, M) over the whole experiment (epoch, E) is kept constant across different groups. This is done by adjusting the Zn concentration of the supplemented diet supplied to compensate for the reduction in the number of days on which Zn-supplemented diet is fed, the latter being spread evenly over the experiment. Idiorrhythms involve offering the diet with n times the overall Zn concentration (M) only every nth day with Zn-deficient diet offered on other days. Idiorrythmic Zn dose-rate feeding changed Zn accumulation in the femur and incisor in a complex bi-modal fashion, indicating that metabolic efficiency of dietary Zn is not constant but depends on Zn dose-rate. In contrast to feeding Zn in the conventional dose-response scheme, iMT and HSP70mRNA were not affected by idiorrhythmic dose-rate feeding. Idiorrhythmic cycling in dietary Zn load posed no risk of a biochemical overload nor caused the animals to be stressed. Idiorrhythmic dose-rate feeding brings the dimension of time to the conventional dose-response model.

Components of the AIN-93 diets as improvements in the AIN-76A diet.

The AIN-93 rodent diets were formulated to substitute for the previous version (AIN-76A) and to improve the performance of animals that consume them. They are called AIN-93G, formulated for growth, and AIN-93M, for maintenance. Major changes included substituting cornstarch for sucrose and soybean oil for corn oil and increasing the amount in order to supply both essential fatty acids (linoleic and linolenic). L-Cystine was substituted for DL-methionine to supplement the casein component. The mineral mix was reformulated to lower the amounts of phosphorus, manganese and chromium, to increase the amount of selenium, and to add molybdenum, silicon, fluoride, nickel, boron, lithium and vanadium. The amounts of vitamins E, K-1 and B-12 were increased over those in the AIN-76A vitamin mix. The AIN-93G diet contains 200 g of casein and 70 g of soybean oil/kg diet. The maintenance diet (AIN-93M) contains 140 g of casein and 40 g of soybean oil/kg diet. The 1993 diets have a better balance of essential nutrients than the 1976 diet and are better choices for studies with laboratory rodents.

Zinc deficiency in adult rats reduces the relative abundance of testis-specific angiotensin-converting enzyme mRNA.

Zinc deficiency results in reduced testicular angiotensin-converting enzyme (ACE) activity and reduced amounts of ACE protein in the testes of young rats. In the present study, we examined the effect of zinc deficiency on the relative abundance of testicular ACE mRNA and its relationship to ACE activity over time. Forty-five male rats at 7 wk of age were placed on one of three feeding regimens: 1) a diet adequate in zinc, 2) a diet deficient in zinc and 3) a diet adequate in zinc that was fed in an amount equal to that consumed by a paired mate fed the zinc-deficient diet. Rats were killed after 3, 5 and 7 wk. Rats fed the zinc-deficient diet had significantly lower (P < 0.01) body weight gain and testis weight at each week sampled than the other groups. They also showed compromised zinc status as evidenced by significantly lower (P < 0.01) serum and testis zinc concentrations. At each period, rats fed the zinc-deficient diet had significantly lower (P < 0.01) testicular ACE activity than rats fed either of the zinc-adequate diets. Coinciding with low ACE activity, there was a lower (P < 0.01) relative abundance of ACE mRNA in the group for the zinc-deficient diet than in either of the zinc-adequate groups. The results suggest that much of the low ACE activity in the testis of rats in the latter stages of zinc deficiency is attributable to a reduction in ACE gene transcription. However, an effect of the deficiency on ACE mRNA turnover is not ruled out.

High zinc concentrations in culture media affect copper uptake and transport in differentiated human colon adenocarcinoma cells.

Previous studies suggest that high dietary zinc reduces the copper status of animals by reducing copper transport across the intestinal mucosa. The present study used an enterocyte mimic, the Caco-2 cell, grown on porus membranes as a model to assess the effects of various concentrations of zinc (6 to 200 micromol/L) in the culture and assay media on 67Cu uptake and transport. Differentiated cells incubated for 7 d in a culture medium containing 50 micromol zinc + 0.7 micromol copper/L transported significantly less 67Cu across the monolayer than similar cells exposed to 6 micromol zinc/L. However, cells exposed to 200 micromol zinc/L for the same period transported significantly more 67Cu than those exposed to 6 micromol zinc/L. Cells exposed for only 1 h to 200 micromol zinc/L in the uptake-transport medium alone did not show lower 67Cu uptake or transport, suggesting that time of exposure of the cells to high zinc was a contributing factor. Caco-2 cells exposed to 50 through 200 micromol zinc/L had higher cellular metallothionein (MT) than those exposed to 6 micromol zinc/L. As the amount of MT in cells increased upon exposure to 50 and 100 micromol zinc/L, the rate of 67Cu transport decreased. At higher zinc concentrations in the medium, there was even more MT in the cells, but a greater rate Of 67Cu transport. These studies demonstrate the use of the Caco-2 cell as a model for copper uptake-transport studies, but the conditions must be rigorously defined and controlled.

Effect of bile/pancreatic secretions on absorption of radioactive or stable zinc. In vivo and in vitro studies.

Biliary/pancreatic (B/P) secretions are a major component of endogenous secretions, and endogenously secreted Zn is a primary means of Zn homeostasis. This study examined whether B/P fluid alters the absorption/reabsorption of Zn and, in doing so, whether this contributes to homeostatic control of Zn. Animal experiments utilized rats fed 10 or 300 micrograms Zn/kg diet. An open-ended gut perfusion study in which 65Zn-labeled B/P fluid or 67Zn-labeled and digested diet found significantly decreased Zn absorption from B/P fluid. Although Zn absorption from both sources was less in animals fed diets higher in Zn, there was no interaction of treatment and diet. Further studies utilizing cultured human colon carcinoma cells (CACO-2) as in vitro models of gut enterocytes found that the presence of B/P fluid significantly decreased Zn retention and/or transport and resulted in a redistribution of cellular Zn after 1200 min of incubation. These studies show that a substance in B/P fluid can decrease the absorption of Zn and also suggest that dietary Zn and Zn associated with B/P secretions are absorbed from distinct pools. However, the lack of an interactive effect with diet, and the amount of time required to see differences in CACO-2 cells, suggest that differences in absorption are not a major contributor to Zn homeostasis.

Amelioration of effects of severe dietary copper deficiency by food restriction in rats.

To test the effect of food restriction on responses to dietary copper deficiency, male weanling rats were fed two amounts of dietary copper via five dietary-intake regimens ranging from ad libitum to 70% of ad libitum intake. Copper-deficient rats exhibited characteristic signs, including depressed organ copper content, reduced activity of copper-dependent enzymes, enlarged hearts, and anemia. Food restriction attenuated the cardiac enlargement, red blood cell defects, and reduction of superoxide dismutase activity in copper-deficient rats. Mineral and enzyme assays suggested that possible mechanisms for this amelioration are the correction of copper status and/or the improvement of antioxidant status. Also, food restriction depressed serum cholesterol and enhanced cytochrome c oxidase activity in both copper-adequate and copper-deficient rats, which compensated for effects of copper deficiency. A second experiment illustrated that the mortality associated with severe copper deficiency was also inhibited by food restriction.

Development and testing of the AIN-93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice.

Because of nutritional and technical problems the AIN-76A rodent diet was revised. One of the new formulations was designated AIN-93G and was suggested for use during growth, pregnancy and lactation studies. Some major differences in this new formulation compared with the AIN-76A diet are as follows: 7 g soybean oil was substituted for 5 g corn oil/100 g diet to increase the amount of linolenic acid; the amounts of vitamins E and K were increased; cornstarch was substituted for most of the sucrose; the amount of phosphorus was reduced to eliminate the problem of nephrocalcinosis in female rats; L-cystine was substituted for DL-methionine; and the manganese concentration was reduced. Various developmental modifications of the AIN-93G diet were fed to weanling rats and mice to determine effects on growth and tissue mineralization. After rats were fed the developmental version of AIN-93G for 16 wk, body weights in both male and female rats were not different from those of rats fed a cereal-based nonpurified diet. After 13 wk, male mice fed this diet weighed 13% more than those fed the nonpurified diet. Body weights of female mice were not affected. The new diet formulation prevented kidney calcification in female rats and mice during 16 wk of feeding.

AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet.

For sixteen years, the American Institute of Nutrition Rodent Diets, AIN-76 and AIN-76A, have been used extensively around the world. Because of numerous nutritional and technical problems encountered with the diet during this period, it was revised. Two new formulations were derived: AIN-93G for growth, pregnancy and lactation, and AIN-93M for adult maintenance. Some major differences in the new formulation of AIN-93G compared with AIN-76A are as follows: 7 g soybean oil/100 g diet was substituted for 5 g corn oil/100 g diet to increase the amount of linolenic acid; cornstarch was substituted for sucrose; the amount of phosphorus was reduced to help eliminate the problem of kidney calcification in female rats; L-cystine was substituted for DL-methionine as the amino acid supplement for casein, known to be deficient in the sulfur amino acids; manganese concentration was lowered to one-fifth the amount in the old diet; the amounts of vitamin E, vitamin K and vitamin B-12 were increased; and molybdenum, silicon, fluoride, nickel, boron, lithium and vanadium were added to the mineral mix. For the AIN-93M maintenance diet, the amount of fat was lowered to 40 g/kg diet from 70 g/kg diet, and the amount of casein to 140 g/kg from 200 g/kg in the AIN-93G diet. Because of a better balance of essential nutrients, the AIN-93 diets may prove to be a better choice than AIN-76A for long-term as well as short-term studies with laboratory rodents.

Zinc deficiency affects the activity and protein concentration of angiotensin-converting enzyme in rat testes.

Zinc (Zn) deficiency causes hypogonadism in a number of different species. Previous work has shown that Zn deficiency reduces the activity of angiotensin-converting enzyme (ACE), a Zn-dependent enzyme, in the testes of prepubertal rats. These studies were designed to determine whether this effect was caused by a change in the concentration of ACE protein. Thirty-five male rats at 26 days of age were divided into three groups. One group was fed ad libitum a Zn-adequate diet (40 mg/kg); another group was fed a similar diet, but deficient in Zn (< 1.0 mg/kg); a third group was pair-fed to the deficient group. After 4 weeks on these regimens, all rats in the ad libitum-fed group and half of the rats in each of the deficient and pair-fed groups were sacrificed, and tissues were collected for analysis. The remaining animals in the Zn-deficient and pair-fed groups were fed a Zn-adequate diet ad libitum for another 2 weeks, then sacrificed. With the use of an enzyme-linked immunosorbent assay for testicular ACE protein, the effect of these treatments on the concentration of ACE protein in testes was determined. After 4 weeks, ACE activity in testes of the Zn-deficient rats was reduced by 74% compared to that in the ad libitum-fed controls. This was accompanied by a 64% reduction in the amount of ACE protein in the testes. There was not a significant effect of pair-feeding. Refeeding Zn-deficient rats a Zn-adequate diet for 2 weeks restored ACE protein concentrations and ACE activity to values not significantly different from those in pair-fed controls. Soluble ACE, but not particulate ACE, of the epididymis was significantly reduced by Zn deficiency. Because the ACE activity of testes has been found primarily in the germinal cells, and soluble ACE in the epididymis is derived from the testes, these findings suggest that the effects of Zn deficiency on testicular and epididymal ACE is caused by an impairment of spermatid development.

Ligands influence Zn transport into cultured endothelial cells.

Experimental results were obtained that demonstrate the importance of the relative concentrations of Zn ligands and their affinities for Zn to the rate of Zn transport across a biological membrane. The transport rate into cultured endothelial cells became saturated near a Zn concentration of 30 microM in the presence or absence of 14% serum. However, the maximum transport rate identified by the saturation plateau was nearly twice as fast from serum-free medium. The addition of histidine or picolinic acid to the medium with serum resulted in the coincidental shift of Zn from high molecular weight ligands to low molecular weight ligands and increased the overall transport rate. In serum-free medium, adding histidine or picolinic acid slowed the transport rate. This indicates that the rate of Zn transport is influenced by the ligand to which it is associated and that altering the relative proportions of specific ligands influences the Zn transport rate. The rate of Zn transport decreased in a stepwise fashion as the albumin to Zn ratio increased from 0 to 4:1, with further increases having little effect. This suggests that albumin has a special role as modulator of Zn transport into endothelial cells. These studies underscore the importance of controlling the relative concentrations of Zn and its ligands in Zn transport kinetic research and suggest that varying their concentrations in a physiological range may be a method of regulating the distribution of Zn into specific cells and tissues.

Zinc transport into endothelial cells is a facilitated process.

The kinetics of zinc transport were examined by measuring the uptake of 65Zn into cultured endothelial cells. This served as a suitable model for characterizing the transport of zinc across a biological membrane (i.e., the plasma membrane). The transport process was saturable under physiological conditions, which indicates a facilitating transport mechanism. Within the physiological range of zinc concentrations, the maximum zinc transport rate was 27 pmoles zinc/(min x mg protein) and it was half maximal at 4.1 microM zinc. Cadmium competitively inhibited zinc transport (Ki = 6.5 microM), while equimolar concentrations of copper and manganese were ineffectual. The rate of zinc transport was substantially reduced at lower temperatures and in the presence of sulfhydryl blockers (sodium iodoacetate and N-ethylmaleimide). Inhibitors of energy metabolism (2,4-dinitrophenol and sodium azide) failed to disrupt zinc transport. These results demonstrate that zinc transport into endothelial cells is a facilitated process (i.e., it is carrier mediated and energy-independent).

Effect of cis-diamminedichloroplatinum (II) on metallothionein induction and trace element metabolism in rats fed different amounts of dietary zinc.

Recent studies have suggested that the induction of metallothionein synthesis in kidneys of mice by the acute administration of bismuth and other trace elements might protect against cis-diamminedichloroplatinum (II) nephrotoxicity. The present study was designed to determine the effects of dietary zinc and cis-diamminedichloroplatinum (II) on the induction of liver and kidney metallothionein and its subsequent effect on nephrotoxicity and trace element metabolism in rats. Male rats were fed diets containing 5, 20, 80, or 320 mg zinc/kg diet for 3 weeks. Each dietary group was subdivided into 3 groups. In one group, each rat received an i.p. injection of 7.5 mg cis-diamminedichloroplatinum (II)/kg b.w. All other rats received saline. During the next three days a second group of rats was pair-fed to the cis-diamminedichloroplatinum (II) injected group. A third group received no treatment and was allowed to eat ad libitum. Results showed that when dietary zinc was increased from 5 mg/kg diet to higher amounts, kidney metallothionein concentration increased twofold. cis-diamminedichloroplatinum (II) treatment increased kidney metallothionein even further, but elevated metallothionein gave no protection from the toxic effects of the drug. Serum copper concentration and ceruloplasmin activity were significantly lower with higher concentrations of dietary zinc, which indicated that these rats were mildly copper-deficient. There was a small but significant depression of superoxide dismutase activity and a highly significant increase in thiobarbituric acid reactive substances in kidneys of rats treated with cis-diamminedichloroplatinum (II) compared to either pair-fed or ad libitum controls. This supports the hypothesis that part of the mechanism for cis-diamminedichloroplatinum (II)-induced toxicity might be caused by free-radical generation. However, the data do not support the hypothesis that metallothionein induction protects the kidney from cis-diamminedichloroplatinum (II) toxicity.