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To understand the potential risks of dietary metals to marine bivalves, it is important to study the interaction between dietary metal bioaccumulation and bivalve feeding behavior. Key processes in affecting the dietary metal influx are the selection of different particles during the ingestion process as well as the differential assimilation of metals during the digestion process. In this study, we quantified the influences of seston quality and quantity on the dietary assimilation of Cd and Zn as well as pre-ingestion particle selectivity in a razor clam Sinonovacula constricta following feeding on radiolabeled diatoms and sediments with different mixtures at four food concentrations. Bioavailability of 109 Cd and 65 Zn from seston was measured by assimilation efficiency (AE) using a pulse-chase feeding technique. The AEs of Cd and Zn were significantly affected by the seston quantity and quality (higher for Zn than they were for Cd and higher for diatoms than for suspended sediments), but were independent of the presence of other particles during the feeding process. Dual gamma radiotracer technique was further employed to study pre-ingestion particle selectivity. Particle selectivity was weak during pre-ingestion in razor clams, although there was evidence that clams might be able to differentiate particles during the process of pseudofeces production. Our study demonstrated that seston composition and quantity substantially affected the bioavailability of Cd and Zn to the razor clams. The results are important to understand the bioaccumulation of metals in clams living in dynamic food environments of estuary., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diet is an important pathway for metal uptake in marine organisms, and assimilation efficiency is one of the most relevant parameters to quantify trophic transfer of metals along aquatic food webs. The most commonly used method to estimate this parameter is pulse-chase feeding using radiolabeled food. This approach is, however, based on several assumptions that are not always tested in an experimental context. The present study aimed to validate the approach by assessing single-feeding and multiple-feeding approaches, using a model species (the turbot Scophthalmus maximus). Using the kinetic data obtained from the single-feeding experiment, the reconstruction of a multi-feeding experiment was tested for consistency with data provided by an actual multi-feeding performed under the same experimental conditions. The results validated the single-feeding approach. Environ Toxicol Chem 2017;36:1227-1234. © 2016 SETAC., (© 2016 SETAC.)

In plant research, radioisotope imaging provides useful information about physiological activities in various tissues and elemental transport between plant organs. To expand the usage of imaging techniques, a new system was developed to visualize beta particles, x-rays and gamma-rays emitted from plant bodies. This real-time radioisotope imaging system (RRIS) visualizes radioactivity after conversion into light with a CsI(Tl) scintillator plate. Herein, the RRIS detection properties of the gamma-ray emitters (22)Na, (65)Zn, (86)Rb, (109)Cd and (137)Cs were evaluated in comparison with those of radioluminography (RLG) using an imaging plate. The lower quantitative detection limit (Bq mm(-2)) during a 15 min period ranged from 0.1 to 4, depending on the nuclide, similar to that of RLG. When the quantitative ability to detect radiation from various Arabidopsis tissues was analyzed, the quantitative capability in silique and the thick internode tended to be low. In an EGS5 simulation, beta particles were the greatest contributors to RRIS imaging of (22)Na, (86)Rb and (137)Cs, and low-energy x-rays contributed significantly to (65)Zn and (109)Cd detection. Thus, both self-absorption and air space between the sample and scintillator surface could impair quantitative RRIS imaging. Despite these issues, RRIS is suggested for quantitative time-course measurements of radionuclide motion within plants.

The transfer characteristics of (137)Cs, (85)Sr and (65)Zn to earthworms (Eisenia andrei) in soils with different amounts of the radionuclides have been investigated. The time-dependent whole-body concentration ratios (CR) were derived for worms in artificially contaminated soils with three different activity concentrations. Two parameters of a first order kinetic model, the equilibrium concentration ratio (CR(eq)) and the effective loss rate constant (k), were estimated by a comparison of experimental CR results with model predictions. The estimated CR(eq) (Bq/kg fresh worm per Bq/kg dry soil) ranged from 3.9 × 10(-4) to 4.1 × 10(-3) for (137)Cs, 1.39 × 10(-3) to 2.94 × 10(-2) for (85)Sr, and 1.39 × 10(-3) to 5.0 × 10(-2) for (65)Zn, and consistently decreased with increasing soil activity concentration but the trend was not statistically significant. The CR(eq) for (137)Cs was one to two orders of magnitude lower than previously reported CR(wo-soil) values (based on field data with much less contaminated soil), that for (85)Sr was comparable with other reported values and for (65)Zn was less two to three orders of magnitude lower than CR(wo-soil) values for stable zinc. The estimated k (d(-1)) values ranged from 9 × 10(-2) to 1.4 × 10(-1) for (137)Cs, 7 × 10(-2) to 2 × 10(-1) for (85)Sr, and 6 × 10(-2) to 1.8 × 10(-1) for (65)Zn, and did not show a relationship with soil activity concentration. The effect of CR(eq) on the total dose rate was insignificant for (137)Cs or (65)Zn because external dose rates to the soil dwelling earthworms due to these radionuclides were much greater than the internal dose rate. In contrast, the total dose from (90)Sr was determined by the internal dose rate and therefore proportional to the CR(eq)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The present study revealed the effects of zinc on the biokinetics of (65)Zn in rats following arsenic intoxication. The animals were segregated into four groups: group I--untreated controls, group II--arsenic treated (100 ppm as NaAsO(2) in drinking water), group III--zinc treated (227 mg ZnSO(4) per liter drinking water), and group IV--arsenic + zinc treated. Each rat was injected intraperitoneally with 1.85 MBq radioactivity of (65)Zn following 3 months of different treatments, and the radioactivity was determined using a suitably shielded scintillation counter. Arsenic treatment showed a significant increase in the fast component (Tb(1)) of the biological half-life of (65)Zn in liver, which remained unaltered in the whole body. Furthermore, arsenic treatment decreased significantly the slow component (Tb(2)) in the whole body, which remained unchanged in the liver. However, zinc supplementation to arsenic-treated rats normalized Tb(1) in the liver, but caused no change in Tb(2) in the whole body. Furthermore, the uptake values of (65)Zn were significantly increased in the liver, brain, kidney, and intestine following arsenic treatment, and the values in the liver and brain were decreased by zinc. Hence, zinc plays a significant role in regulating the biokinetics of (65)Zn in the liver and the whole body of arsenic-intoxicated rats.

Dietary factors are considered crucial for the prevention of initiating events in the multistep progression of colon carcinoma. There is substantial evidence that zinc may play a pivotal role in host defense against several malignancies, including colon cancer. The present study was conducted to evaluate the kinetics of (65)Zn utilization following experimental colon carcinogenesis in rat model. Twenty rats were segregated into two groups viz., untreated control and dimethylhydrazine (DMH) treated. Colon carcinogenesis was established through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 16 weeks. Whole body (65)Zn kinetics followed two compartment kinetics, with Tb(1) representing the initial fast component of the biological half-life and Tb(2), the slower component. The present study revealed a significant depression in the Tb(1) and Tb(2) components of (65)Zn in DMH treated rats. Further, DMH treatment caused a significant increase in the percent uptake values of (65)Zn in the colon, small intestine, kidney and blood, whereas a significant decrease was observed in the liver. Subcellular distribution revealed a significant increase in (65)Zn uptake in the mitochondrial and microsomal fractions following 16 weeks of DMH supplementation. In conclusion, the present study demonstrated a slow mobilization of (65)Zn during promotion of experimentally induced colon carcinogenesis and provides a physiological basis for the role of (65)Zn in colon tumorigenesis, which may have clinical implications in the management of colon cancer.

The adsorption of pair nuclides 60Co/Co and 65Zn/Zn by solid phase and their availability to plants were studied in soil suspension conditions and in the sod podzolic soil under controlled moisture. The situation when the radionuclide in constant activity is entered to system (surface water, soil) with different heavy metal contamination has been modeled. Was observed that soil contamination with heavy metal Co (Zn) significantly reduce sorption of the radionuclide 60Co (65Zn) by solid phase. As a result, the activity concentration of the 60Co or 65Zn in soil solution and, therefore, their mobility and potential availability to plants increases in 2-4 times with the total metal concentration increasing. The difference between two elements is that high Co concentration increase root uptake of the 60Co, whereas soil contamination with Zn reduce activity of 65Zn in the 14-days barley plants that may reflects diverse plant necessity of two elements and more important role of the isotopic exchange effect in the case of Zn.

Isotopic dilution with stable zinc and chelation with DTPA are recommended for removal of radioactive zinc from the body; however, it is unclear which method is more effective. In the present study, the efficacies of these methods were compared in order to determine which treatment should be selected in case of internal contamination with radioactive zinc. Intraperitoneal administration of stable zinc dose-dependently removed 65Zn from the mouse body. However, the dose could not be increased above 3 mg/kg due to its toxicity. Oral administration of zinc was less effective than intraperitoneal administration at the same dose. Our results suggest that the recommended dose of stable zinc (2-3 mg/kg, p.o.) has little efficacy. The efficacies of Ca-DTPA and Zn-DTPA were strongly dependent on the elapsed time after 65Zn exposure. Zn-DTPA was more effective than Ca-DTPA, and its recommended dose (30 micromol/kg) significantly removed 65Zn. Therefore, chelation therapy with Zn-DTPA should be started as soon as possible after internal contamination with radiozinc.

Arabidopsis halleri is a well-known zinc (Zn) hyperaccumulator, but its status as a cadmium (Cd) hyperaccumulator is less certain. Here, we investigated whether A. halleri can hyperaccumulate Cd and whether Cd is transported via the Zn pathway. Growth and Cd and Zn uptake were determined in hydroponic experiments with different Cd and Zn concentrations. Short-term uptake and root-to-shoot transport were measured with radioactive 109Cd and 65Zn labelling. A. halleri accumulated > 1000 mg Cd kg(-1) in shoot dry weight at external Cd concentrations >or= 5 microm, but the short-term uptake rate of 109Cd was much lower than that of 65Zn. Zinc inhibited short-term 109Cd uptake kinetics and root-to-shoot translocation, as well as long-term Cd accumulation in shoots. Uptake of 109Cd and 65Zn were up-regulated, respectively, by low iron (Fe) or Zn status. A. halleri was much less tolerant to Cd than to Zn. We conclude that A. halleri is able to hyperaccumulate Cd partly, at least, through the Zn pathway, but the mechanisms responsible for cellular Zn tolerance cannot detoxify Cd effectively.

The crustacean hepatopancreas is an epithelial-lined, multifunctional organ that, among other activities, regulates the flow of calcium into and out of the animal's body throughout the life cycle. Transepithelial calcium flow across this epithelial cell layer occurs by the combination of calcium channels and cation exchangers at the apical pole of the cell and by an ATP-dependent, calcium ATPase in conjunction with a calcium channel and an Na+/Ca2+ antiporter in the basolateral cell region. The roles of intracellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum (ER) in transepithelial calcium transport or in transient calcium sequestration are unclear, but may be involved in transferring cytosolic calcium from one cell pole to the other. The ER membrane has a complement of ATP-dependent calcium ATPases (SERCA) and calcium channels that regulate the uptake and possible transfer of calcium through this organelle during periods of intense calcium fluxes across the epithelium as a whole. This investigation characterized the mechanisms of calcium transport by lobster hepatopancreatic ER vesicles and the effects of drugs and heavy metals on them. Kinetic constants for 45Ca2+ influx under control conditions were K(n) (m)=10.38+/-1.01 microM, J(max)=14.75+/-1.27 pmol/mg protein x sec, and n=2.53+/-0.46. The Hill coefficient for 45Ca2+ influx under control conditions, approximating 2, suggests that approximately two calcium ions were transported for each transport cycle in the absence of ATP or the inhibitors. Addition of 1 mM ATP to the incubation medium significantly (P<0.01) elevated the rate of 45Ca2+ influx at all calcium activities used and retained the sigmoidal nature of the transport relationship. The kinetic constants for 45Ca2+ influx in the presence of 1 mM ATP were K(n) (m)=12.76+/-0.91 microM, J(max)=25.46+/-1.45 pmol/mg protein x sec, and n=1.95+/-0.15. Kinetic analyses of ER 65Zn2+ influx resulted in a sigmoidal relationship between transport rate and zinc activity under control conditions (K(n) (m)=38.63+/-0.52 microM, J(max)=19.35+/-0.17 pmol/mg protein x sec, n=1.81+/-0.03). The Addition of 1 mM ATP enhanced 65Zn2+ influx at each zinc activity, but maintained the overall sigmoidal nature of the kinetic relationship. The kinetic constants for zinc influx in the presence of 1 mM ATP were K(n) (m)=34.59+/-2.31 microM, J(max)=26.09+/-1.17 pmol/mg protein x sec, and n=1.96+/-0.17. Both sigmoidal and ATP-dependent calcium and zinc influxes by ER vesicles were reduced in the presence of thapsigargin and vanadate. This investigation found that lobster hepatopancreatic ER exhibited a thapsigargin- and vanadate-inhibited, SERCA-like, calcium ATPase. This transporter displayed cooperative calcium transport kinetics (Hill coefficient, n approximately 2.0) and was inhibited by the heavy metals zinc and copper, suggesting that the metals may reduce the binding and transport of calcium when they are present in the cytosol.

Whole body counting studies of 65Zn indicated that the Tb1 (the faster component) was significantly decreased while the slower component (Tb2) was increased significantly following ethanol treatment. Interestingly, following zinc treatment to ethanol treated rats, slower component (Tb2) of 65Zn came back to within normal limits while the faster component (Tb1) got significantly elevated in comparison to ethanol treatment. Percent uptake values of 65Zn were found to be increased in liver, intestine, muscle, brain and kidney, and decreased in bone under alcoholic conditions. Interestingly, the uptake values of 65Zn in all the organs except muscle were reverted back to within normal limits upon zinc supplementation to these ethanol intoxicated animals. A significant decrease in zinc contents was noticed in ethanol treated rats, which, however, were raised to normal levels upon zinc supplementation: Copper levels, on the other hand, were significantly enhanced in both ethanol fed and combined ethanol + zinc treated rats. Calcium levels were significantly decreased in both ethanol and zinc treated rats, which however were further reduced upon zinc supplementation to ethanol fed rats. However, no significant change was observed in the concentrations of sodium and potassium in any of the treatment groups. In conclusion, zinc appears to play a protective role by normalizing the turnover of 65Zn in whole body as well as in its uptake in different organs under alcoholic conditions.

The tubular intestine of the American lobster Homarus americanus was isolated in vitro and perfused with a physiological saline whose composition was based on hemolymph ion concentrations and contained variable concentrations of (3)H-l-histidine, (3)H-glycyl-sarcosine and (65)Zn(2+). Mucosa to serosa (M-->S) flux of each radiolabelled substrate was measured by the rate of isotope appearance in the physiological saline bathing the tissue on the serosal surface. Addition of 1-50 micromol l(-1) zinc to the luminal solution containing 1-50 micromol l(-1) (3)H-l-histidine significantly (P<0.01) increased M-->S flux of amino acid compared to controls lacking the metal. The kinetics of M-->S (3)H-l-histidine flux in the absence of zinc followed Michaelis-Menten kinetics (K(m)=6.2+/-0.8 micromol l(-1); J(max) =0.09+/-0.004 pmol cm(-2) min(-1)). Addition of 20 micromol l(-1) zinc to the luminal perfusate increased both kinetic constants (K(m)=19+/-3 micromol l(-1); J(max)=0.28+/-0.02 pmol cm(-2) min(-1)). Addition of both 20 micromol l(-1) zinc and 100 micromol l(-1) l-leucine abolished the stimulatory effect of the metal alone (K(m)=4.5+/-1.7 micromol l(-1); J(max)=0.08+/-0.008 pmol cm(-2) min(-1)). In the absence of l-histidine, M-->S flux of (65)Zn(2+) also followed the Michaelis-Menten relationship and addition of l-histidine to the perfusate significantly (P<0.01) increased both kinetic constants. Addition of either 50 micromol l(-1) Cu(+) or Cu(2+) and 20 micromol l(-1) l-histidine simultaneously abolished the stimulatory effect of l-histidine alone on transmural (65)Zn(2+) transport. Zinc-stimulation of M-->S (3)H-l-histidine flux was significantly (P<0.01) reduced by the addition of 100 micromol l(-1) glycyl-sarcosine to the perfusate, as a result of the dipeptide significantly (P<0.01) reducing both l-histidine transport K(m) and J(max). Transmural transport of (3)H-glycyl-sarcosine was unaffected by the presence of either l-histidine or l-leucine when either amino acid was added to the perfusate alone, but at least a 50% reduction in peptide transport was observed when zinc and either of the amino acids were added simultaneously. These results show that (3)H-l-histidine and (65)Zn(2+) are cotransported across the lobster intestine by a dipeptide carrier protein that binds both substrates in a bis-complex (Zn-[His](2)) resembling the normal dipeptide substrate. In addition, the transmural transports of both substrates may also occur by uncharacterized carrier processes that are independent of one another and appear relatively specific to the solutes used in this study.

This study was designed to determine the effect of zinc on the biological half-lives of 65Zn in whole body and liver and on distribution of 65Zn in different organs of rats following nickel toxicity. Sprague-Dawley (SD) rats received either nickel in the form NiSO4.6H2O at a dose of 800 mg/L in drinking water, zinc in the form of ZnSO4.7H2O at a dose of 227 mg/L in drinking water, and nickel plus zinc or drinking water alone for a total duration of 8 wk. All of the rats were injected with a tracer dose of 0.37 MBq 65Zn at the end of the treatment period. The effects of different treatments were studied on biological half-lives of 65Zn in whole body and liver and on the distribution of 65Zn in different organs of rats. In the present study, we have noted that nickel treatment to normal rats caused a significant decrease in the slow component (Tb2) in liver, which improved following zinc supplementation. Nickel administration to normal-diet-fed animals caused significant lowering in the percentage uptake of 65Zn values in the brain, liver, and intestine. However, the administration of zinc to nickel-treated rats improved the status of 65Zn in different organs. The Tb2 in the liver and the percentage uptake of 65Zn values elevated following zinc supplementation to nickel-treated rats.

On the basis of the previous evidence that 65Zn concentrations in the brain of EL (epilepsy) mice was affected by induction of seizures, 65Zn movement in the brain was quantitatively evaluated in ddY mice treated with kainate. Six days after intravenous injection of 65ZnCl2, mice were intraperitoneally injected with kainate (10 mg/kg x 6 times in 2 weeks). Myoclonic jerks were observed during treatment with kainate. Twenty days after 65Zn injection, 65Zn distribution in the brain was compared between the kainite-treated and control mice. 65Zn distribution in the brain of the kainate-treated mice was overall lower than in the control mice. 65Zn concentration was significantly decreased in the frontal cortex, hippocampal CA1, thalamus and hypothalamus by treatment with kainate. These results demonstrate that kainate-induced seizures are linked to decreased zinc concentrations in the brain.

Zn efficiency (ZE) is the ability of plants to maintain high yield under Zn-deficiency stress in the soil. Two bean ( Phaseolus vulgaris L.) genotypes that differed in ZE, Voyager (Zn-efficient) and Avanti (Zn-inefficient), were used for this investigation. Plants were grown under controlled-environment conditions in chelate-buffered nutrient solution where Zn(2+) activities were controlled at low (0.1 pM) or sufficient (150 pM) levels. To investigate the relative contribution of the root versus the shoot to ZE, observations of Zn-deficiency symptoms in reciprocal grafts of the two genotypes were made. After growth under low-Zn conditions, plants of nongrafted Avanti, self-grafted Avanti and reciprocal grafts that had the Avanti shoot scion exhibited Zn-deficiency symptoms. However nongrafted and self-grafted Voyager, as well as reciprocal grafts with the Voyager shoot scion, were healthy with no visible Zn-deficiency symptoms under the same growth conditions. More detailed investigations into putative shoot-localized ZE mechanisms involved determinations of leaf biomass production and Zn accumulation, measurements of subcellular Zn compartmentation, activities of two Zn-requiring enzymes, carbonic anhydrase and Cu/Zn-dependent superoxide dismutase (Co/ZnSOD), as well as the non-Zn-requiring enzyme nitrate reductase. There were no differences in shoot tissue Zn concentrations between the Zn-inefficient and Zn-efficient genotypes grown under the low-Zn conditions where differences in ZE were exhibited. Shoot Zn compartmentation was investigated using radiotracer ((65)Zn) efflux analysis and suggested that the Zn-efficient genotype maintains higher cytoplasmic Zn concentrations and less Zn in the leaf-cell vacuole, compared to leaves from the Zn-inefficient genotype under Zn deficiency. Analysis of Zn-requiring enzymes in bean leaves revealed that the Zn-efficient genotype maintains significantly higher levels of carbonic anhydrase and Cu/ZnSOD activity under Zn deficiency. While these data are not sufficient to allow us to determine the specific mechanisms underlying ZE, they certainly point to the shoot as a key site where ZE mechanisms are functioning, and could involve processes associated with Zn compartmentation and biochemical Zn utilization.

Vacuolar compartmentalization or cell wall binding in leaves could play a major role in hyperaccumulation of heavy metals. However, little is known about the physiology of intracellular cadmium (Cd) sequestration in plants. We investigated the role of the leaf cells in allocating metal in hyperaccumulating plants by measuring short-term (109)Cd and (65)Zn uptake in mesophyll protoplasts of Thlaspi caerulescens "Ganges" and Arabidopsis halleri, both hyperaccumulators of zinc (Zn) and Cd, and T. caerulescens "Prayon," accumulating Cd at a lower degree. The effects of low temperature, several divalent cations, and pre-exposure of the plants to metals were investigated. There was no significant difference between the Michaelis-Menten kinetic constants of the three plants. It indicates that differences in metal uptake cannot be explained by different constitutive transport capacities at the leaf protoplast level and that plasma and vacuole membranes of mesophyll cells are not responsible for the differences observed in heavy metal allocation. This suggests the existence of regulation mechanisms before the plasma membrane of leaf mesophyll protoplasts. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of "Ganges," whereas it decreased Cd accumulation in A. halleri protoplasts, indicating that Cd-permeable transport proteins are differentially regulated. The experiment with competitors has shown that probably more than one single transport system is carrying Cd in parallel into the cell and that in T. caerulescens "Prayon," Cd could be transported by a Zn and Ca pathway, whereas in "Ganges," Cd could be transported mainly by other pathways.

Zinc is an essential element, which can act as a neuromodulator and also is bound in zinc proteins in the brain. The olfactory bulb contains high concentrations of zinc. In the present study, 65Zn(2+) was applied on the olfactory epithelium of rats and pikes and the transport of the metal in the olfactory system was then examined. Administration of 65Zn(2+) in the nasal cavity of rats or the olfactory chambers in pikes resulted in an uptake of the metal in the olfactory epithelium and a transport of the metal along the primary olfactory neurons to their terminations in the olfactory bulbs. Low levels of 65Zn(2+) passed these terminals and continued into the interior of the bulbs. In the rats 65Zn(2+) was also detected in the anterior parts of the olfactory cortex. Subcellular fractionations of the olfactory mucosa and olfactory bulb of rats given 65Zn(2+) intranasally showed that the metal is bound both to particulate cellular constituents and to cytosolic components in these tissues. Gel chromatography indicated that some of the zinc in the cytosol is bound to metallothionein in the olfactory mucosa and bulb. Inhalation of zinc-containing dusts or fumes occurs in some work-places and may imply high exposure of the nasal tissues. It is not known whether neurotoxicity may be related to uptake of zinc in the olfactory system. However, this is an issue which deserves attention, since zinc dysregulation has been implied to play a role in Alzheimer's disease. In addition, impairment of the sense of smell and degenerative changes of the olfactory tissues have been seen in early stages of some neurodegenerative disorders.

The development of zinc deficiency in adults was studied in a metabolism experiment involving 31 adult, female rats labeled homogenously with 65Zn. The animals were fed restricted amounts (8 g/day) of a semisynthetic diet containing either 58 microgram Zn/g (control, n = 7) or 2 microgram Zn/g (Zn deficiency, n = 24). Control animals were sacrificed at day 0 (n = 3) and day 29 (n = 4). Zinc deficient animals were sacrificed at day 1, 2, 4, 7, 11, 16, 22, and 29 (3 animals per group). The development of zinc deficiency comprised 4 phases: (I) Fecal Zn excretion needed several days to adjust to the low level of Zn intake. The high initial Zn loss via feces was counterbalanced mainly by Zn mobilization from the skeleton. (II) During the 2nd week of deficiency Zn mobilization from tissue storage changed transiently to soft tissues (mainly muscle and fat tissue). (III) After the 2nd week the skeleton resumed to mobilize Zn. (IV) At the end of the study the skeleton Zn storage was exhausted and alkaline phosphatase activity indicated severe Zn deficiency. Urinary Zn excretion was too small to contribute quantitatively to changes in Zn metabolism during any phase of Zn deficiency. In conclusion, adults may compensate a deficient Zn supply by mobilizing tissue Zn for several weeks: The skeleton revealed to be the major short-term as well as long-term source of whole body tissue Zn that can be mobilized.

A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described. Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn. Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC). However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC). To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC. This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM. Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM. To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope. The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.

The nutritional bioavailability of zinc from cooked milled, undermilled, and brown Philippine rice (variety PSB Rc14) was evaluated in rats, comparing results based on weight gain, tibia zinc incorporation (slope ratio analyses), and zinc radiotracer retention. Milling reduced the phytic acid and mineral content of the rice, resulting in zinc concentrations of 16.5, 19.4, and 27.2 microg/g and phytate/zinc molar ratios of 4, 20, and 28 for milled, undermilled, and brown rice, respectively. Measured zinc bioavailability was similar whether using growth, bone zinc, or radioisotope retention as criteria, at approximately 92, 86, and 77% of zinc sulfate, for milled, undermilled, and brown rice, respectively. However, the higher percent bioavailability of the zinc after milling was insufficient to compensate for the lower zinc content. With respect to zinc, the nutritional value was inversely related to milling, providing approximately 15, 17, and 21 microg bioavailable zinc/g rice, respectively, for milled, undermilled and brown rice of this variety.

Following the works of Velazquez et al. (1999), Jo-Seung et al. (2000), Wang et al. (2001), Danscher et al. (2001) and the criteria of Zinc-containing neurons established by Frederickson et al.(2000), we have found the presence and localisation of Zinc in the neurons of the dorsal root ganglia of Wistar rat, by using Timm's thecnique and by studying the autoradiographic uptake of 65Zn. The agreement between the results of both techniques allows us to classify these spinal ganglion neurons as Zinc-containing neurons and also, to confirm some of the results of Velazquez et al. (1999).

DNA polymerase III (pol III) of Gram-positive eubacteria is a catalytically bifunctional DNA polymerase:3'-5' exonuclease [Low, R. L., Rashbaum, S. A., and Cozzarelli, N. R. (1976) J. Biol.Chem. 251, 1311-1325]. The pol III protein conserves, between its exonuclease and dNTP binding sites, a 35-residue segment of primary structure with the potential to form a zinc finger-like structure [Berg, J. M. (1990) Ann. Rev. Biochem. 19, 405-421]. This paper describes results of experiments which probe the capacity of this segment to bind zinc and the role of this segment in enzyme function. The results of metal and mutational analysis of a model pol III derived from Bacillus subtilis indicate that (i) the Gram-positive pol III is a metalloprotein containing tightly bound zinc in a stoichiometry of 1, (ii) the zinc atom is bound within the 35-residue segment, likely in one of two probable finger-like structures, and (iii) the integrity of the zinc-bound structure is specifically critical to the formation and/or function of the enzyme's polymerase site.

The effects of ascorbic acid, sodium citrate, and sodium bicarbonate on 59Fe-transferrin, 54Mn-transferrin, and 65Zn-transferrin uptake by the receptors disposed of plasma membrane isolated from lactating mouse mammary gland cells have been investigated. The effect of 10(-2) mol/L ascorbic acid alone and in combination with NaHCO3 on the 59Fe-transferrin uptake is significant and positive. 54Mn-transferrin and 65Zn-transferrin binding to the cell receptors are influenced optimally by 0.5 mol/L sodium bicarbonate. Sodium citrate alone or in combination with other substances always has a negative effect on binding of these three metals. It is suggested that a precise mechanism may exist with large possibilities to rearrange metal uptake and its transport from blood to milk.

The absorption of Zn or Fe from breakfast meals containing oat porridge prepared from malted and soaked oats and a control porridge made from untreated oats was measured in human subjects. The effect on Zn and Fe absorption of reducing the phytate content of oat-porridge meals was examined in each subject by extrinsic labelling of porridge with 65Zn and of bread rolls with 55Fe and 59Fe, and measuring whole-body retention and the erythrocyte uptake of isotopes. Each experiment comprised nine to ten subjects. The absorption of Zn from malted-oat porridge with a phytate (inositol hexaphosphate) content of 107 mumol was 18.3%, and significantly higher (P < 0.05) than from the control porridge containing 432 mumol phytate (11.8%). Fe absorption from the meal containing malted-oat porridge with 107 mumol phytate (Expt 2) was also significantly improved (P < 0.05) compared with that from the meal containing control porridge with 437 mumol phytate. The average increase in Fe absorption was 47%, or from 4.4 to 6.0%. In the breakfast meal containing malted porridge with 198 mumol phytate (Expt 3) the increase in Fe absorption was not significantly improved. Even though the phytate content was reduced to a greater extent in Expt 3 than Expt 2, the average increase in Fe absorption in Expt 3 was only 25% more than that from the meal containing control porridge (with 599 mumol phytate), depending on the higher absolute amount of phytate. In conclusion, an improvement in Zn and Fe absorption from oat products can be achieved by practising malting and soaking in the processing of oats. This may be of importance in the prevention of mineral deficiency in vulnerable groups.

The primary structure of human interleukin-3 contains two amino acid consensus sequences at Glutamate 22- Histidine 26 and Histidine 95-Histidine 98, that are characteristic for zinc binding proteins. Therefore, the hypothesis was tested that human interleukin-3 binds zinc specifically by either one or both sequences. Protein dotblotting, followed by probing with radioactive zinc demonstrated specific zinc binding of interleukin-3. Metal specificity was confirmed by competition experiments with 12 other divalent- and trivalent metal ions. Protease treatment combined with plasma desorption mass spectrometry was used to localize the zinc binding domain. Specific zinc binding was restricted to a fragment composed of Threonine 11-Lysine 28 and Asparagine 80-Lysine 100. It was found to decrease by a factor of five when either of these two amino acid stretches was missing. It is concluded that human interleukin-3 is a zinc binding protein. Interleukin-3 zinc binding capacity is largely determined by both moieties of the protein that contain the consensus sequences. In addition we propose that the zinc binding of hIL-3 is involved in (de)phosphorylation of the hIL-3 receptor.

1. A significant proportion of plasma zinc exists complexed with amino acids. The effect of amino acids on the accumulation of radioactive zinc by rat erythrocytes was studied in vitro, to investigate the hypothesis that zinc might be transported into cells as an amino acid-zinc complex. 2. L-Histidine (500 microM-10 mM) stimulated 65Zn uptake; 50 mM-L-histidine gave a slight inhibition of uptake. D-Histidine (500 microM-10 mM) inhibited uptake in a dose-dependent manner. A non-zinc-binding amino acid, L-alanine, did not affect 65Zn uptake. 3. The effect of L-histidine was sodium dependent and temperature dependent, but was DIDS insensitive. These properties suggest that zinc is being transported as a zinc-histidine complex, utilizing an amino acid carrier system. Uptake of zinc in the presence of L-histidine differed from the previously described ionic mechanism, and may represent a physiological route of uptake. 4. L-Histidine stimulated efflux of 65Zn from pre-loaded cells. 5. The relevance of transport of a zinc-histidine complex is discussed with reference to histidinaemia, and as a significant zinc transport system in the presence of the very low ionic zinc concentrations found in plasma.

Mice were gavaged with zinc-65 solution, 8.6-19.3 kBq per mouse, and the whole-body retention and organ content of zinc-65 were measured at different times after administration. The age-dependence of the fractional absorption of zinc-65 from the gastrointestinal tract (f1), the endogenous faecal excretion fraction of zinc-65 (EFEF), tissue distribution and whole-body retention were determined. The f1 values obtained were 0.86 +/- 0.15, 0.64 +/- 0.11, 0.52 +/- 0.07 and 0.39 +/- 0.02 in suckling, adolescent, young adult and older mice, respectively. The EFEF values determined were 0.083 +/- 0.008, 0.099 +/- 0.004, 0.122 +/- 0.018 and 0.144 +/- 0.005 of intraperitoneally injected zinc-65 in suckling, adolescent, young adult and older mice at administration. Zinc-65 mainly distributed in the liver, muscle, lung, kidneys and bone. In some tissues, there was an inverse relationship between the relative content of gavaged zinc-65 and the animal's age at administration. The whole-body biological half-lives of zinc-65 increased with animal age. The influence of the age-dependent variation of zinc-65 metabolism on internal dose and on radiation protection is discussed.

The isolated trophoblast from human placenta at term has been used as model for placental transport. Binding to the cells showed to be of low specificity and very limited (less than 10%) in presence of serum. Uptake of zinc was 90% reduced at 4 degrees C and depended on the presence of sulfhydryl groups (as suggested with the inhibition produced by N-ethylmaleimide). There is considerable efflux (50%) of zinc from preloaded cells and the process was enhanced by presence of histidine in the medium. The model proved to be useful in studying the mode of zinc transport. The results indicate that the uptake of zinc by the trophoblast is probably the limiting process in the placental transport of this metal.

We present 64Cu uptake studies in cultured muscle cells from a one-year-old patient with Menkes' disease. The cultured muscle cells from the patient showed a five-fold higher 64Cu uptake than control muscle cells. Copper uptake in muscle cells was of the same magnitude as that found in fibroblasts from the patient and also from other Menkes' patients. The copper content of a muscle biopsy from the patient was twice that of a control biopsy. The enhanced uptake is probably copper specific, since zinc uptake was unaltered in both muscle cells and fibroblasts from the patient. Cytochrome c oxidase in the muscle of the patient was reduced to one-third of the value for controls, which is in agreement with the hypothesis that in Menkes' disease copper accumulates in a biologically non-active form. However, in cultured muscle cells and fibroblasts from the patient the cytochrome c oxidase activity was in the normal range, probably because of the relatively large amount of copper already available in the culture medium.

Alkaline phosphatase from Pseudomonas fluorescens has been partially purified. Labelled 65ZnCl2 in the culture medium is incorporated in the most purified preparations. The enzyme shows a pH optimum of 7.2-7.5 and a Km value for p-nitrophenyl phosphate of 1 times 10(-4). Zinc ions at high concentration inhibit enzyme activity. The protective effect of Mg on the inactivation of alkaline phosphatase by heat is also reported.

A transplantable, metastasizing prostatic adenocarcinoma (Tumor I) in Lobund Wistar rats was examined for activity and distribution of five hydrolytic enzymes and for ability to accumulate radioactive zinc. The results suggest that the tumor had arisen in the ventral lobe of the prostate and that its growth was not affected by orchiectomy, adrenalectomy, or replacement treatment with exogenous androgen or corticosteroids. The androgen independency of the tumor was further shown by the low uptake of 3H-testosterone, in contrast to the high uptake in the ventral prostate. Tumor growth was retarded by Cytoxan but not by 5-fluorouracil, Estracyt, or streptozotocin, three agents clinically effective in the treatment of some patients with prostatic cancer resistant to endocrine therapy. It is concluded that this tumor in Lobund Wistar rats may be an adequate model for human prostatic cancers resistant to the agents mentioned above.

Despite studies by several investigators of human gastrointestinal 65Zn absorption, implications of these data for evaluation of functional zinc status are unclear because limited numbers of normal subjects have been studied. To evaluated zinc absorption in normal humans, 75 subjects (31 women, 44 men, ages 18 to 84 yr) were given 10 micro Ci carrier-free 65Zn orally after an overnight fast. Absorption calculated from total body retention measured 7, 14, and 21 days after administration of tracer was 65 +/- 11% (mean +/- 1 SD), range from 40 to 86%. Comparison of these results with those for patients with a variety of diseases indicate that patients exhibit a wider range of absorption and, in four of six studies patients exhibit decreased mean zinc absorption. These results of gastrointestinal zinc absorption in a large number of normal humans offer a basis for a clearer comparison with data from patients who exhibit abnormalities of zinc absorption.

The use of stable isotopes in a study on zinc bioavailability was evaluated in rats by comparing the zinc absorption of a stable isotope 70Zn and a radioisotope 65Zn simultaneously. The rats of low zinc status were obtained by feeding 1.2% phytic acid from sodium phytate and 12 ppm zinc from zinc sulfate in a semipurified diet for 14 days. The rats were then randomly assigned into five groups of ten rats. The rats were intubated with a test diet mixture with a phytate:zinc molar ratio of 0, 6.25, 12.5, 25 or 50 along with one microCi of 65Zn and 0.02 mg of 70Zn in 2.5 ml of intubating solution. Four hours after the intubation, rats were allowed to consume the diets having the same phytate:zinc ratio as used for intubation ad libitum. Feces were collected every 24 hours for a total of 168 hours. The 65Zn and 70Zn content of diets and fecal samples were determined and the percent of zinc absorption was calculated. The results of this study indicated that the zinc absorption determined by these two isotopes were highly correlated with a correlation coefficient ratio of 0.914.

65Zinc absorption in patients suffering from generalized scleroderma was studied by means of whole-body counting technique following a single dose of 65Zn. In 4 untreated patients the mean 65Zn absorption was calculated to 35% (range 20-59%). Five patients receiving oral D-penicillamine had a numerically higher mean absorption value of 55% (range 37-74%). The results corroborate earlier studies on the effect of D-penicillamine on 65Zn absorption in rats.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.

The regional concentrations of nine radiochemicals were measured in rat brain after induction of cerebral ischemia to identify tracers concentrated by brain undergoing selective neuronal necrosis. Transient (30 minute) forebrain ischemia was produced in the rat; 24 hours after cerebral recirculation the radiochemicals were injected intravenously and allowed to circulate for 5 hours. The brain concentrations of the radiochemicals in dissected regions were determined by scintillation counting. Forebrain ischemia of this nature will produce extensive injury to striatal neurons but will spare the great majority of neocortical neurons at 24 hours. The regional concentrations of these radiochemicals varied considerably in both control and ischemic animals. In postischemic animals, 4 radionuclides (63Ni, 99TcO4, 22Na, and [3H]tetracycline) were concentrated in the irreversibly damaged striatum in amounts ranging from 1.4 to 2.4 times greater than in normal tissue. The concentrations of 65Zn, 59Fe, 32PO4, and 147Pm in postischemic brain were similar to or less than those in normal brain. The concentration of [14C]EDTA was increased in injured and uninjured brain of postischemic rats. Autoradiographic analysis of the distribution patterns of some of these ions in normal animals showed that 99TcO4, 22Na, 65Zn, and 59Fe were distributed more uniformly throughout the brain than were 32PO4, 63Ni, and 147Pm. At 24 or 48 hours after ischemia, 63Ni, 99TcO4, and 22Na were preferentially concentrated in the damaged striatum and hippocampus, whereas 65Zn, 59Fe, 32PO4, and 147Pm did not accumulate in irreversibly injured tissue. Of the radiochemicals tested to date, Ni, TcO4, and tetracycline may be useful for diagnosing ischemic brain injury in humans, using positron emission tomography.

Adult and neonatal rats and neonatal pigs were gavaged with solutions of metal radionuclides to determine gastrointestinal absorption. Zinc-65 and technetium-95m were well-absorbed by both age groups; chromium-51, cadmium-109, tin-113, promethium-147, and plutonium-238 were not. The quantities of the poorly absorbed metals that were absorbed by neonates were between 4 and 100 times higher than those absorbed by adult rats. Autoradiograms prepared from the entire small intestine of the neonatal rat showed that 109Cd was retained in the duodenum. In contrast, measurements in the piglets showed much higher 109Cd retention in the ileum than in the duodenum. Autoradiograms and radiochemical measurements of 147Pm and 238Pu in both neonatal rats and swine showed the highest level of retention in the ileum. The results indicate that, for most of the metals studied, absorption from the gastrointestinal tract is substantially higher for neonatal than for adult rats.

Analysis of bone retention data using 2- or 3-compartment models gives nearly equal relative accretion rates for zinc and calcium (85Sr) in endochondral bone and calcifying cartilage. The rate for zinc in cortical bone is 70% of that for calcium. As the existence in cortical bone of large, slowly exchanging compartments limitating accretion is consistent with the data, the relative accretion rate for zinc may more closely reflect the relative accretion rate of cortical bone.

Uptake of 65Zn and distribution of 65Zn, total zinc, and insulin were measured in rat islets and islet granules under different conditions of islet culture. Specific activity of islet zinc (65Zn/zinc) was less than 15% that of extracellular zinc even after 48 h. In contrast, once in the islet, 65Zn approached 70% of equilibrium with granular zinc in 24 h and apparent equilibrium by 48 h. During a 24-h culture, at either high or low glucose, reduction of both islet zinc and insulin occurred. However, zinc depletion was greater than that predicted if zinc loss was proportional to insulin depletion and occurred only from the granular compartment, which represents only one third of the total islet zinc. Extension of culture to 48 h caused additional insulin depletion, but islet zinc was unchanged. Omission of calcium during the 48-h culture caused a predicted increase in insulin retention, presumably by inhibiting secretion; however, zinc retention was not increased proportionately. Pretreatment of rats with tolbutamide caused a massive depletion of insulin stored in isolated islets, with little change in total islet zinc; subsequent culture of these islets resulted in a greater loss of granular zinc than predicted from the small loss of granular insulin. None of the conditions tested affected the percentage of either 65Zn or total zinc that was distributed in the islet granules. Results show that zinc exists in a metabolically labile islet compartment(s) as well as in secretory granules; and extra-granular zinc, although not directly associated with insulin storage, may act as a reservoir for granular zinc and may regulate insulin synthesis, storage, and secretion in ways as yet unknown.

Estracyt (estramustine phosphate) injected intraperitoneally, 100 mg, per Kg. three days a week for four weeks, retarded growth of the R-3327 tumor in intact rats and in orchiectomized rats given androgen. The growth inhibition was accomplished by reduction of tumor deoxyribonucleic acid concentration and of the activities of acid phosphatase, leucine aminopeptidase, and other hydrolases. Histologic examination revealed cellular necrosis particularly prominent in the orchiectomized, androgen-treated rats. Estracyt did not affect the uptake of 65-Zn in the tumors but markedly reduced the high uptake in the dorsolateral prostate. There was no accumulation of 3H or 14C in the tumors after intravenous administration of 3H, 14C-labeled Estracyt, but the isotope concentrations decreased much in the same way as they decreased in the dorsolateral prostate. The isotopes were retained in the ventral prostate, where their concentrations were approximately twenty times higher than those in the muscle four hours after injection. The results demonstrate the value of the R-3327 tumor in the evaluation of drugs of potential clinical use for the treatment of prostatic cancer. The results also show that Estracyt has an antitumor effect which is not dependent on the antigonadotropic action of the drug.

The effects of tetracycline on the absorption of orally ingested 65Zn was studied on rats by whole body counting assay. 65Zn was given as a single dose to groups of rats, five in each, which were started on tetracycline. Tetracycline was given in daily doses of 25 mg, 50 mg, 100 mg, and 200 mg for 10 days. From the 6th day of the study, 65Zn retention when plotted against time in a semilog plot, approximated linearity. The net absorption of 65Zn in the various groups was determined by extrapolating to zero time the linear curve segment of individual retention curves. Except for the lowest tetracycline dosage, tetracycline significantly impaired 65Zn absorption. The elimination rate of retained 65Zn from the 6th day was significantly higher in the group receiving 200 mg tetracycline as compared with the control group.