Philip G. Reeves

Bioavailability as an issue in risk assessment and management of food cadmium: A review

The bioavailability of cadmium (Cd) from food is an important determinant of the potential risk of this toxic element. This review summarizes the effects of marginal deficiencies of the essential nutrients zinc (Zn), iron (Fe), and calcium (Ca) on the enhancement of absorption and organ accumulation and retention of dietary Cd in laboratory animals. These marginal deficiencies enhanced Cd absorption as much as ten-fold from diets containing low Cd concentrations similar to that consumed by some human populations, indicating that people who are nutritionally marginal with respect to Zn, Fe, and Ca are at higher risk of Cd disease than those who are nutritionally adequate. Results from these studies also suggest that the bioavailability of Cd is different for different food sources. This has implications for the design of food safety rules for Cd in that if the dietary source plays such a significant role in the risk of Cd, then different foods would require different Cd limits. Lastly, the importance of food-level exposures of Cd and other potentially toxic elements in the study of risk assessment are emphasized. Most foods contain low concentrations of Cd that are poorly absorbed, and it is neither relevant nor practical to use toxic doses of Cd in experimental diets to study food Cd risks. A more comprehensive understanding of the biochemistry involved in the bioavailability of Cd from foods would help resolve food safety questions and provide the support for a badly needed advance in international policies regarding Cd in crops and foods.

Adaptation responses in rats to long-term feeding of high-zinc diets : emphasis on intestinal metallothionein

Abstract When faced with an abundance of heavy metals in the diet, higher animals produce metallothionein (MT) in the intestinal mucosa to sequester the metals in an attempt to reduce their absorption into the body. This paper shows that this strategy may only be used for short-term exposure, and a more effective, sustainable strategy is adopted during long-term exposure. The concentration of dietary zinc was abruptly elevated and offered to rats continually for a specified period. The results showed that an abrupt change in dietary zinc caused an immediate elevation of intestinal MT concentration, which remained elevated for about 2 weeks and then began to decline. After about 5 weeks, MT concentrations in the intestines of rats fed high-zinc diets were not different from controls fed normal-zinc diets for the entire period. The concentrations of zinc in serum, liver, and kidney followed the same course as mucosal MT, elevated during the initial phase of feeding high-zinc and near control values during the latter phase of feeding. Although the rats were consuming diets with zinc concentrations about 7 fold higher than controls, and intestinal MT concentrations were not elevated, the serum and tissue concentrations of zinc were near control values. This suggests that the induction of intestinal MT may be an immediate and short-term strategy for coping with high intakes of certain metals, and that some other, more efficient mechanism is adopted during long-term exposure.

Ligands Influence Zn Transport into Cultured Endothelial Cells

Abstract 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 μM 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.

Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 &mgr;mol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using (65)Zn. Similar cells were labeled with (65)Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 &mgr;mol Zn/L was slower (p < 0.05) than that in cells exposed to 5 &mgr;mol Zn/L. The AP to BL transport rate in the 25 &mgr;mol Zn/L group was only 40% (p < 0.05) of that in the 5 &mgr;M group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 &mgr;mol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 &mgr;mol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 &mgr;mol Zn/L group than in the 5 &mgr;M group, but the rate of BL Zn release was higher in cells treated with 25 &mgr;mol Zn/L than in those treated with 5 &mgr;mol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.

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 µM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D3 plus Interferon-γ (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-myrlstate 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 hosts defense system.

Zinc uptake and transcellular movement by CACO-2 cells: Studies with media containing fetal bovine serum

A series of experiments using CACO-2 cells, have been conducted to describe the cellular events occurring during Zn uptake and transcellular movement in the presence of media containing fetal bovine serum (FBS). CACO-2 cells were grown for 21 days on either T-25 cell culture flasks or semipermeable membrane inserts maintained in six-well culture plates. Experiments were then conducted using normal growth medium containing FBS, to which 65Zn was added. The rate of Zn transport in an apical to basolateral direction was much greater than the rate in the opposite direction. To study whether uptake and movement exhibited saturation kinetics, different concentrations of Zn (1–200 μ m ) were added with 65Zn. Uptake at the basolateral membrane was saturable; apical to basolateral movement, basolateral to apical movement, and uptake at the apical membrane were not. 65Zn moved apically to basolaterally at a small but constant rate regardless of the basolateral concentration of Zn. The binding ligands for Zn in the cytosol differed depending on the prior Zn status of the cell and the time postlabeling. Endogenous 65Zn was released to the apical and basolateral sides at different rates. These data confirm a previous report that uptake and transcellular movement of Zn is different at the apical and basolateral membrane, and they further show that the presence of Zn-binding ligands alters these processes.

Copper metabolism in metallothionein-null mice fed a high-zinc diet

Abstract Humans and animals develop low copper (Cu) status when fed diets containing large amounts of zinc (Zn) for an extended period. Current theory states that Zn-induced metallothionein (MT) in the intestinal mucosa binds Cu and prevents its absorption. We tested this theory by using a mouse model with a disruption in the MT gene that renders it incapable of producing functional MT-I and MT-II (MT-null). If the theory is true, then the MT-null mouse should not develop low Cu status when fed a high Zn diet. For 1 week, groups of 4-week-old MT-null and control mice were fed a diet that contained 35 mg Zn and

Zinc-induced metallothionein and copper metabolism in intestinal mucosa, liver, and kidney of rats

Abstract Large doses of parenteral zinc (Zn) and/or the feeding of high Zn diets to animals or humans for long periods affects copper (Cu) metabolism. Previous work suggests that Zn-induced metallothionein (MT) in intestinal epithelial cells binds Cu and inhibits its absorption. This study was designed to determine the effects of treating rats with high dietary or high parenteral Zn on Cu metabolism and its relationship to MT in the intestinal epithelium, liver and kidney. Six-week-old male rats were fed for one week a control diet containing 42 mg Zn and 6 mg Cu/kg. They were then divided into three groups. One group continued to receive the control diet while another received a similar diet containing 560 mg Zn/kg. A third group, fed the control diet, received a subcutaneous dose of 90 mg Zn/kg body weight every 2–3 days for the duration of the experiment. Rats from each group were killed on days 7 and 14. Low Cu status in Zn-treated rats was indicated by lower than normal serum Cu concentration, serum ceruloplasmin activity, low liver and kidney Cu concentrations and low cytochrome C oxidase activity. None of these changes, however, were related to an increase in Cu as a result of Zn-induced MT in the intestinal epithelial cell. Instead, as the MT concentrations rose, Cu concentration decreased. This study suggests that the effects of high Zn treatment on Cu status are not the result of the long-held theory that Zn-induced intestinal MT sequesters Cu and prevents its passage to the circulation. Instead, it may be caused by a direct effect of high lumenal Zn concentrations on Cu transport into the epithelial cell.

Zinc Deficiency Affects the Activity and Protein Concentration of Angiotensin-Converting Enzyme in Rat Testes

Abstract 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 libitumfed 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.

Zinc-and/or cadmium-induced intestinal metallothionein and copper metabolism in adult rats☆

Abstract Feeding diets with high zinc or cadmium concentrations to animals compromises their copper status. Previous work suggested that metallothionein (MT), induced in the intestinal mucosa by zinc or cadmium, binds copper and inhibits its absorption. More recent studies showed that this mechanism may not be operative for zinc and that intestinal MT adapts to long-term feeding of high-zinc diets. The present study was designed to determine the effects of feeding high-zinc and/or high-cadmium diets on the induction of intestinal MT and its subsequent effect on copper status. Six-week-old male rats were placed in a 2 × 2 × 3 factorial experiment with two concentrations of dietary zinc (60 and 350 mg/kg), two concentrations of dietary copper (3 and 9 mg/kg), and three concentrations of dietary cadmium (0, 1, and 5 mg/kg). After 3 weeks, the difference in the MT concentration between rats fed high- and normal-zinc diets was only 1.5 times. However, rats fed the highest amount of cadmium had MT concentrations about five time higher than those not fed cadmium. In both the zinc and cadmium groups, the concentration of intestinal zinc and cadmium followed that of MT; however, the copper concentrations were not changed. Although intestinal MT was not elevated appreciably in zinc-fed rats, the copper status of these rats fed 3 mg of copper/kg of diet was severely depressed. Rats fed 9 mg of copper/kg of diet were not affected. The copper status in rats fed high-cadmium and 3 mg of copper/kg of diet was depressed even more than with a high-zinc diet. This study suggests that the effects of high dietary zinc or cadmium on copper status are not the result of induced intestinal MT binding of copper thus preventing its passage into the circulation.