65Zn Injection Research Articles

Elimination of zinc-65 from the brain under kainate-induced seizures.

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.

Role of zinc released by stimulation in rat amygdala

The movement and role of actively functioning zinc, i.e., vesicular zinc, in the amygdala was studied, based on the data that 65Zn is localized in the limbic system, which may correspond to the regions with high densities of zinc-containing neuron terminals. When release of 65Zn into the extracellular space was examined 2 hr or 25 hr after injection of 65ZnCl2 into the amygdala, 65Zn release was facilitated by stimulation with high K+ 2 hr after injection, but not 25 hr after injection. Even 25 hr after 65Zn injection into the amygdala, approximately 95% of total 65Zn in the brain was detected in the injected area. These results suggest that zinc released into the extracellular space in the amygdala is not readily restored to the presynaptic vesicles. Moreover, to chelate zinc in the extracellular space (and in the synaptic vesicles) in the amygdala, the amygdalae were perfused with 10 μM diethyldithiocarbamate during behavioral tests for odor recognition. The olfactory sensation was temporarily disturbed by the perfusion. These results suggest that vesicular zinc is essential to the function of the amygdala, e.g., olfactory function. J. Neurosci. Res. 57:405–410, 1999. © 1999 Wiley-Liss, Inc.

Role of zinc released by stimulation in rat amygdala

The movement and role of actively functioning zinc, i.e., vesicular zinc, in the amygdala was studied, based on the data that 65Zn is localized in the limbic system, which may correspond to the regions with high densities of zinc-containing neuron terminals. When release of 65Zn into the extracellular space was examined 2 hr or 25 hr after injection of 65ZnCl2 into the amygdala, 65Zn release was facilitated by stimulation with high K+ 2 hr after injection, but not 25 hr after injection. Even 25 hr after 65Zn injection into the amygdala, approximately 95% of total 65Zn in the brain was detected in the injected area. These results suggest that zinc released into the extracellular space in the amygdala is not readily restored to the presynaptic vesicles. Moreover, to chelate zinc in the extracellular space (and in the synaptic vesicles) in the amygdala, the amygdalae were perfused with 10 microM diethyldithiocarbamate during behavioral tests for odor recognition. The olfactory sensation was temporarily disturbed by the perfusion. These results suggest that vesicular zinc is essential to the function of the amygdala, e.g., olfactory function.

Zinc transport from the striatum and substantia nigra

65Zn was unilaterally injected into the striatum or substantia nigra of rats to see the transport of intracerebral zinc (Zn). In the case of intrastriatal injection, 65Zn was densely distributed in the ipsilateral medial forebrain bundle (MFB) and the substantia nigra. On unilateral colchicine injection into the MFB, 65Zn distribution in the ipsilateral substantia nigra decreased significantly compared to that of the contralateral one after 65Zn injection into the bilateral striata. These results suggest the presence of axonal transport of 65Zn taken up by striatonigral γ-aminobutyric acid (GABA)-ergic and/or nigro-striatal dopaminergic neurons. On the other hand, in the case of intranigral injection, 65Zn was distributed in the ipsilateral MFB, striatum, globus pallidus, pontine reticular nuclei, and pontine nuclei. 65Zn distribution in the pons 1 day after intranigral injection was very similar to that 6 days after intrastriatal injection, suggesting that, in the case of intrastriatal injection of 65Zn, nigral 65Zn, which was transported anterogradely and/or retrogradely from the striatum, was transported to the postsynaptic neurons through the synapse and then transported to the pons.

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine.

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway, 65Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When 65ZnCl2 was intravenously injected 4 days after treatment with 6-OHDA, 65Zn was concentrated in the ipsilateral substantia nigra 6 days after 65Zn injection. On the other hand, 19 d after treatment with 6-OHDA, 65Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH4(99)TcO4, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA, 99Tc was concentrated in the ipsilateral substantia nigra 30 min after 99Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.

Zinc transport in the rat olfactory system

To study zinc (Zn) mobility in the rat olfactory tract, brain distribution of 65Zn after injection into the olfactory bulb or amygdaloid nuclei was analyzed by autoradiography. Twenty-four hours after 65Zn injection into the olfactory bulb, 65Zn was distributed in the ipsilateral piriform cortex, amygdaloid nuclei and the anterior commissure. Moreover, in the case of injection of a higher level of 65Zn into the olfactory bulb, 65Zn was distributed in the ipsilateral entorhinal cortex in addition to the above regions. Twenty-four hours after 65Zn injection into the amygdaloid nuclei, 65Zn was distributed in the ipsilateral piriform and entorhinal cortex. These results suggest that Zn is intraneuronally transported along the olfactory tract. Zn may be taken up by the piriform neurons after release from the secondary olfactory neuron terminals and transported to the entorhinal area.

Relative influence of amount of dietary zinc and infusion of protein into the duodenum on the amount of zinc in biliary/pancreatic secretions

A series of studies were undertaken to determine the relative influence of dietary Zn vs the presence of protein in the duodenum on the excretion of Zn in biliary/pancreatic fluid (B/P) of rats. Animals were fed diets ranging from 10 to 300 mg Zn/kg diet for 7 weeks and then fasted and cannulated for bile or B/P fluid collection. Secretions of fasted rats were collected 0.5 hr and then egg white was infused into the gut and stimulated secretions were collected. Elevated dietary Zn slightly increased Zn and decreased Cu content of B/P fluid but did not alter protein concentration or activity of carboxypeptidase A or B. In contrast, duodenal stimulation greatly increased Zn and protein content and activity of carboxypeptidase A and B. In a separate experiment, animals fed 10 or 300 mg Zn/kg diet were similarly cannulated and infused with protein at intervals from 6 to 240 hr following injection of 65Zn. Dietary Zn slightly elevated B/P Zn but did not alter biliary Zn concentration. Maximal biliary Zn output occurred 6 hr after injection whereas B/P Zn output was maximal after 12 hr. These results suggest that the Zn in B/P secretions of rats is there primarily as a result of metabolic demand by the pancreas, and these secretions are not a major means of maintaining Zn homeostasis.

The Murine Mutation, Lethal Milk, Results in Production of Zinc-Deficient Milk

Normal or mutant pups that nurse dams homozygous for the lethal milk (lm) mutation die as a result of zinc deficiency. Previous determinations of the zinc concentration of the mutant milk have been conflicting. This work demonstrates that the amount of 65Zn recovered in the organs of pups following in intraperitoneal injection of 65Zn to lactating dams was reduced 50-60% in the stomach, 25-30% in the gut and 50-75% in the blood and carcass, for both normal and mutant pups nursing mutant dams, relative to pups nursing normal dams. For pups nursing mutant dams, the zinc concentration of the stomach and contents was determined by atomic absorption spectrometry and found to be 144 nmol/g wet wt (for mutant pups) and 147 nmol/g wet wt (for normal pups). Pups nursing normal dams had stomach zinc concentrations of 322 nmol/g wet wt (mutant pups) and 312 nmol/g wet wt (normal pups). Administration of an oral dose of 65Zn (27 kBq) to normal and mutant adult mice showed that after 24 h there was no significant difference in the distribution of 65Zn in the liver, gut, lung, spleen, kidney, brain, skin, blood, pancreas or heart or in the carcass. We conclude that the major effect of the lethal milk mutation is the production of Zn-deficient milk.

Effects of histidine on tissue zinc distribution in rats.

Histidine has been reported to affect body zinc status by increasing urinary zinc excretion. The effects of experimental histidinemia on distribution of 65Zn in anesthetized rats were studied. Infusion of L-histidine at a rate sufficient to raise plasma concentrations to approximately 2 mM for 6 h starting 48 h after a single intraperitoneal 65Zn injection did not alter 65Zn activities in a variety of tissues when compared with anesthetized uninfused animals. However, plasma 65Zn and erythrocyte 65Zn were decreased, and liver 65Zn was increased. If 65Zn was injected intravenously during histidine infusion, net accumulation of zinc by some tissues was increased, but uptake by others was reduced relative to uninfused animals. In all cases, however, uptake expressed relative to plasma 65Zn levels was increased when allowance was made for the more rapid fall in plasma 65Zn during histidine infusion. Similar infusions of D-histidine produced quantitatively similar effects. Since enzymatic mechanisms and amino acid carriers would be expected to show stereoselectivity, such processes are unlikely to be involved in the zinc distribution changes described. The possibility of zinc transport by a hitherto unidentified carrier is discussed. These experiments confirm that histidinemia can affect zinc status, but any associated changes in urinary zinc excretion do not seem adequate to account for the tissue changes found.

γ線亜致死線量照射マウスへの亜鉛，マンガンおよびマグネシウムの多量投与による死亡率の低減

Radioprotective effect has been observed in mice which have subcutaneously received a single dose of abundant inorganic zinc or manganese 24 hours before gamma-ray irradiation with a sublethal dose. Mechanism of the radioprotection has been suggested to be an abundant production of metallothionein (MT) induced by the metals. But process of the protection might be more complicated because immunological and hematopoietic functions are remarkably inhibited by the irradiation and many zinc binding-compounds with biological functions are known at present. Furthermore, the effect of these metals has been observed by the administration only before the irradiation. Radioprotective effect was shown in mice which were administered with not only zinc and manganese, but also magnesium. It seems that mechanism of the radioprotection of magnesium is different from that of zinc or manganese since magnesium is not known to induce MT production. It was observed that the relative concentration ratio of radioactivity in tissues and organs at 7th day (after injection of 65Zn) showed a marked difference between in irradiated and non-irradiated mice.

High molecular weight zinc- and copper-binding proteins in the liver of the rat; Effect of centrifugation

After intravenous injection of 65Zn or 64Cu in the rat, the presence of high molecular weight Zn- and Cu-binding proteins in the cytosol of the liver was investigated, using gel chromatography over Sephacryl S-300. About 10% of the 65Zn was found in the void volume of the column, viz., in a fraction with a molecular weight of > 500 000 D. A similar percentage of the cytoplasmic 64Cu turned out to be associated with these proteins. Little change was seen in this distribution during the time interval of 0.25 to 8 h post injection. About the same percentage of non- radioactive Zn and Cu was found in this fraction. This high molecular weight fraction was sedimented when the liver homogenate was centrifuged for a prolonged time and/or at high g-value. It is different from the high molecular weight artifacts produced by, e.g., oxidation. The results stress the need for clearly specifying the conditions of centrifugation in descriptions of experiments.

Intestinal Absorption and Excretion of Zinc in Streptozotocin-Diabetic Rats as Affected by Dietary Zinc and Protein

65Zn was used to examine the effects of dietary zinc and protein on true zinc absorption and intestinal excretion of endogenous zinc by an isotope dilution technique in streptozotocin-diabetic and control rats. Four groups each of diabetic and control rats were fed diets containing 20 ppm Zn, 20% egg white protein (HMHP); 20 ppm Zn, 10% egg white protein (HMLP); 10 ppm Zn, 20% egg white protein (LMHP); and 10 ppm Zn, 10% egg white protein (LMLP). Measurement of zinc balance was begun 9 d after an i.m. injection of 65Zn. True zinc absorption and the contribution of endogenous zinc to fecal zinc excretion were calculated from the isotopically labeled and unlabeled zinc in the feces, duodenum and kidney. Results from the isotope dilution study indicated that diabetic rats, but not control rats, absorbed more zinc from 20 ppm zinc diets than from 10 ppm zinc diets and that all rats absorbed more zinc from 20% protein diets than from 10% protein diets. Furthermore, all rats excreted more endogenous zinc from their intestines when dietary zinc and protein levels resulted in greater zinc absorption. In diabetic and control rats, consuming equivalent amounts of zinc, the amount of zinc absorbed was not significantly different, but the amount of zinc excreted by the intestine was less in the diabetic rats. Decreased intestinal excretion of endogenous zinc may be a homeostatic response to the increased urinary excretion of endogenous zinc in the diabetic rats and may also lead to the elevated zinc concentrations observed in some organs of the diabetic rats.

65Zn turnover in short-term alloxan diabetic rats.

65Zn was injected subcutaneously in eleven rats with alloxan diabetes of five days duration and in nine control rats. Fifty hours after the injection of 65Zn the animals were killed with an overdose of ether. In the alloxan diabetic rats signs of an altered zinc metabolism were found with an increased excretion of zinc in urine and possibly also faeces and an elevated zinc concentration in the liver compared to controls. The results also indicate an altered distribution of serum zinc in alloxan treated rats. Future studies will be necessary to demonstrate whether changes in zinc metabolism are secondary to the onset of diabetes or a primary effect of alloxan treatment.

ORGAN DISTRIBUTION OF 65ZN IN EHRLICH ASCITES CANCER-BEARING MICE

Organ distribution of 65Zn injected i.p. or s.c. into Ehrlich ascites cancer-bearing mice was examined with the following results.1) Distribution of 65Zn injected i.p. was examined as a function of time (1-24 hr). 65Zn was found mainly in ascites cancer cells reaching a maximum (26%) 6 hr after the injection and its concentration was gradually decreased thereafter. Radioactivity of 65Zn in liver, kidney, pancreas and spleen were less than that in cancer cells. Disappearance of the radioactivity contained in cancer cells was very little after washing.2) Subcellular distribution pattern of 65Zn in ascites cancer cells was examined extending from 1 to 24 hr after i.p. injection. The amount of 65Zn in supernatant fraction was highest followed by nuclear, microsomal and mitochondrial fractions in the descending order and the above distribution pattern was same at any times.3) Radioactivity of 65Zn administered s.c. into cancer-bearing mice was significantly increased in the liver and decreased in pancreas and blood with no changes in the kidney and spleen compared with that in normal mice. After the i.p. injection of 65Zn into cancer-bearing mice, 66% of total radioactivity was found in ascites and 65Zn concentration in the liver, kidney, pancreas, spleen and blood were less than that in normal mice.4) When the radioactivity of 65Zn injected i.p. or s.c. in the subcellular fractions of liver and cancer cells are expressed per mg protein, the highest concentration of 65Zn in the liver was found in the supernatant fraction followed by microsomal, nuclear and mitochondrial fractions in the decending order. These distribution pattern of 65Zn was equal with that of cancer cells.5) 65Zn in the liver and cancer cells binds loosely with protein. After the treatment of each subcellular fractions obtained from liver and cancer cells with (NH4) 2SO4 (70% saturation) and trichloroacetic acid (10% in final), most of 65Zn was distributed in supernatant excepting the liver nuclear fraction in which most of radioactivity exists in precipitate by (NH4) 2SO4.6) Subcellular distribution of 65Zn in mammary carcinoma was examined 24 hr after the s. c. injection. 65Zn concentration is equal in nuclear and supernatnat fractions followed by microsomal and mitochondrial fractions in the descending order. The distribution pattern of 65Zn in nuclear fraction was different from that in the liver.

Effect of Zinc Deficiency and Restricted Feeding on Protein and Ribonucleic Acid Metabolism of Rat Brain

Male weanling rats fed an egg albumin diet deficient in zinc (0.2 ppm Zn) for 3 weeks gained very little weight. During a 6-hour period following intraperitoneal injection of 65Zn, the brains of the zinc-deficient animals took up a larger percentage of the injected dose than did the brains of animals fed the zinc-supplemented diet (75 ppm Zn) either ad libitum, or restricted to allow a weight gain comparable to the zinc-deficient rats. The in vivo incorporation of intracerebrally injected 14C-L-leucine and 3H-uridine into the trichloroacetic acid-precipitable protein and the ribonucleic acid (RNA), respectively, of brain was determined. Neither zinc deficiency nor restricted feeding affected the rate of incorporation of 3H-uridine into RNA of the brain. The relative specific activities of the proteins isolated from the zinc-deficient and restricted-fed animals were somewhat similar, and were lower than those from the zinc-supplemented rats fed ad libitum.

Dialysis Studies of Liver Zinc in Zinc-deficient and Control Rats

Experiments were designed to study possible effects of zinc deficiency on zinc binding by rat liver. Either 3 hours after intravenous 65Zn injection or after 10 days of feeding 65Zn-containing diets, low zinc (-Zn) and zinc-supplemented (+Zn) rats were killed and liver homogenates dialyzed against 8 successive volumes of buffered 10-3M EDTA, CDTA, histidine, cysteine, glycine, or buffer alone. Based on the dialysis results, liver zinc was delineated into 4 pools. When 65Zn was fed for 10 days to label zinc pools uniformly, dietary zinc deficiency had no significant effect on the amount of 65Zn removed by any of the dialyzing agents, which suggests that the binding pattern of liver zinc, like that of total zinc concentration, is not altered in zinc deficiency. When 65Zn was injected intravenously, the percentage of liver 65Zn removable at 3 hours postinjection by EDTA, but not by histidine, was significantly smaller in -Zn than in +Zn rats. This suggests that the rate of incorporation of zinc into certain liver pools is altered in zinc deficiency. Explanations for this effect were suggested, based on the difference in growth rate between the +Zn and -Zn rats and on the possible increase in the turnover rate of a firmly bound zinc pool in zinc deficiency.

Effects of Chelation and of Various Cations on the Mobility of Foliar-Applied 65zn in Subterranean Clover

Injection of 65Zn of high specific activity into old leaves of subterranean clover (Trifolium 8ubterraneum L.) resulted in the virtual immobilization of the isotope in the laminae of the treated leaves. The addition of 30, 300, or 500 p.g respectively of non-radioactive zinc to the dose resulted in progressively increased ,movement of 65Zn into other parts of the plant.