The dithizone,Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS

Summary From rats intravitally treated with dithizone (diphenyl-thiocarbazone) brains and spinal cords were removed and freeze-dried. The dithizonates present in the CNS tissue were extracted with carbon tetrachloride and subjected to a multielement analysis (proton activation, PIXE). It was found that the extract contained two metals. Most of the metal was zinc, but small traces of copper were also dectected. Because prior treatment with the chelating agent, dithizone, can block both the Timm and the selenium metal staining methods, it is suggested that the three techniques label predominantly zine in the neuropil (DTS-zine).     

Damaging action of dithizone on insulin-releasing cells

Alterative action of dithizone was investigated in the experiments on various species of animals (fish, frogs, pigeons, mice, guinea pigs, golden hamsters, rats, rabbits, cats and dogs). The data received support the previously advanced suggestion that unsaturated (electrophilic) zinc complex formation is the basic mechanism of the alterative chelant's action.     

Selective staining and disintegration of intestinal eosinophilic granule cells in Atlantic salmon after intraperitoneal injection of the zinc chelator dithizone

Following intraperitoneal injection of the zinc ion chelator dithizone into Atlantic salmon Salmo salar , staining and morphological changes in intestinal eosinophiic granule cells (EGC) were observed in both cryo-preserved and chemical-preserved tissue sections. In cryo sections rapid and selective vital staining of EGC was observed within 5 min of dithizone injection. Intensely stained red granules appeared as a result of the formation of a complex between dithizone and zinc ions. Stained EGC appeared on both sides of the stratum compactum as well as in the lamina propria . Stained EGC started to disintegrate 5 min after the injection of dithizone, and little of the zinc-dithizone complex was observed 15 h later. The disappearance of the stain coincided with an intense degranulation and disintegration of EGC. Fifteen hours after dithizone treatment regenerating cells were observed in Masson's trichrome stained sections. Four to five days after injection, a fully regenerated continuous EGC layer was observed. Simultaneously with the disintegration of EGC an increase in plasma lysozyme activity occurred. EGC resembles mammalian Paneth cells in their possession of lysozymecontaining granules and their staining by, and response to, dithizone. EGC may represent a central component of zinc metabolism in Atlantic salmon.     

Zinc contents of pancreatic islets in animals of various species after administration of diabetogenic agent, dithizone]

A decrease of zinc content in pancreatic islet was shown in animals which received diabetogenic agent dithizone. The changes of islet zinc content occurred mainly on account of insulin-producing cells and in coincidence with glycemia changes.     

Depletion of metal in the rat hippocampal mossy fibre system by intravital chelation with dithizone

Summary Adult albino rats were given dithizone by repeated intraperitoneal injections followed by a standard test dose. After a standard interval of 15 or 60 minutes the animals were killed by vascular perfusion with a buffered acidified formaldehyde solution. The brains were immediately removed, frozen, and cut at a constant thickness of 160 microns on a freezing microtome.The red stain of metal dithizonate known to be particularly marked in the hippocampal mossy fibre system was observed and recorded by a technique based on photography of sections. It was found that dithizone in doses of 25 mg per kg body weight, injected hourly for 12 hours prevented the dithizone staining of the mossy fibre system caused by the test dose in control animals. The result is interpreted as evidence of true depletion of metal.This study was supported in part by U.S.P.H.S. Grant NS 07998. This aid is gratefully acknowledged. Einar Hansen, Thorkild Nielsen, Birgit Örum, Albert Meier, Karin Sörensen and Inger Madsen rendered valuable technical assistance.     

Evidence from dithizone and selenium zinc histochemistry that perivascular mossy fiber boutons stain preferentially "in vivo"

This paper describes a perivascular staining pattern that is obtained when dithizone or sodium selenite are used to label zinc intravitally. Our observations indicate that the perivascular staining is a result of zinc labeling in mossy fiber boutons adjacent to capillaries and suggest that there might be a special blood brain barrier in the mossy fiber regions.     

Evidence from dithizone and selenium zinc histochemistry that perivascular mossy fiber boutons stain preferentially “in vivo”

Summary This paper describes a perivascular staining pattern that is obtained when dithizone or sodium selenite are used to label zinc intravitally. Our observations indicate that the perivascular staining is a result of zinc labeling in mossy fiber boutons adjacent to capillaries and suggest that there might be a special blood brain barrier in the mossy fiber regions.     

Translocation of zinc from vacuole to nucleus during yeast meiosis

A novel staining procedure employing the UV fluorochrome DAPI (4',6-diamidino-2-phenylindole X 2HCl) and dithizone (diphenylthiocarbazone) was developed for microcytochemical determination of sites of zinc localization in Saccharomyces cerevisiae Hansen. In vegetative cells vacuolar polyphosphate bodies stained with dithizone, whereas in sporulating cells nucleoli and centriolar plaques were dithizone-positive. Hence, dithizone not only permitted localization of zinc but also indicated zinc translocation from vacuolar to nuclear compartments during differentiation from the vegetative to sporulated state.     

A simple,cheap but reliable method for evaluation of zinc chelating properties

BackgroundZinc is an essential trace element. Both its lack and excess are associated with pathological states. The former is more common and can ensue from the excessive treatment with clinically used iron/copper chelators.Aim and methodThe aim of this work was to prepare a reliable, rapid and cheap method for the screening of zinc chelation. Spectrophotometric assessment using a known zinc indicator dithizone was selected.ResultsInitial screening performed by comparison of spectra of dithizone and its complex with zinc suggested 530 and 570 nm as suitable wavelengths for determination of zinc at pH 4.5 while 540 and 590 nm for pH 5.5–7.5. Additional research showed the lower wavelengths to be more suitable. The sensitivity of the method was always bellow 1 μM with good linearity relationship between absorbance and zinc concentration. The method suitability was confirmed by use of two known zinc chelators, ethylenediaminetetraacetic acid (EDTA) and N,N,N′,N′-tetrakis(2-pyridylmethyl)-1,2-ethylenediamine (TPEN).ConclusionThis method represents a sufficiently precise method for zinc chelation screening usable at pathophysiologically relevant pH conditions. Such method can be employed for both screening of novel zinc chelators and for testing affinity of other metal chelators for zinc.     

Effects of dithizone on the electroencephalogram recorded from the mouse hippocampus in vivo

In the present study the author examined the effects of dithizone on hippocampal and cortical EEG by power spectral analysis in the moving mouse. Following results were obtained. Administration of dithizone 100 mg/kg i. p. produced almost loss of electrical activities on EEG which began 409 sec after injection and lasted approximately up to 706 sec. In recovery period waveform showed shift to slower frequencies apparent by 60 min. Heart rate decreases were seen between 5 and 20 min after 100 mg/kg i. p. injection. Dithizone produced dose-dependent changes in hippocampal and heart rate activities. Abolished EEG by dithizone administration were immediately recovered by zinc-acetate application. Injection of vehicle had no significant effect on hippocampal and cortical EEG.     

Autometallographic localization of protein-bound copper and zinc in the common winkle,Littorina littorea: A light microscopical study

Summary Copper (Cu), zinc (Zn) and calcium (Ca) were demonstrated histochemically by means of conventional stains (rubeanic acid for copper, dithizone for zinc, and cobalt nitrare for calcium) and by autometallography in various tissues of winkles (Littorina littorea) sublethally exposed to either copper or zinc dissolved in sea water. Rubeanic acid and dithizone procedures exhibited poor sensitivity: there was no positive reaction after fixation tissues with Bouin's fixative, and only a weak reaction after ethanol fixation. Autometallography, however, produced a positive reaction with both fixatives in the form of black silver deposits in some key cell types. In winkles not exposed to either copper nor zinc, autometallographically demonstrated metals were found in the connective tissue pore cells, the lysosomes of digestive cells, the basal lamina of the digestive tubule epithelium, and cytoplasmic granules in the epithelial cells of the stomach wall. In addition, in winkles exposed to copper, metal deposits were present in some apical cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, and the circulating haemocytes. In winkles exposed to zinc, metal deposits were found in the basal cytoplasmic granules of ciliated cells in the gill epithelium, the mucous secretion of gill mucocytes, the apex and basal lamina of the nephrocytes in the kidney, and the connective tissue layer surrounding the blood vessels. Additionally, calcium was demonstrated histochemically in the cytoplasm of digestive cells, the cytoplasm of the epithelial cells of the stomach wall, the mucocytes of gills, the basal lamina of the kidneys, the haemocytes, the calcium and pore cells of connective tissue, and the oocyte cytoplasm. Metals were not detected by any procedure in sperm cells, in the cytoplasmic granules of oocytes, or in the basophilic cells in the digestive tubules. In conclusion, autometallography is a highly sensitive method and provides an excellent tool to localize protein-bound copper and zinc in molluscan tissues, and its use in combination with conventional histochemical or chemical methods is highly recommended.     

Heavy metals in the spinal cord of normal rats and of animals treated with chelating agents: a quantitative (zinc, copper, and lead) and histochemical study

The amounts of zinc, copper, and lead in the rat spinal cord were determined by means of flameless atomic absorption spectrophotometry. Zinc was present in a concentration about 100 p.p.m. (dry weight), copper in a concentration about 5 p.p.m., and lead in slightly more than 1 p.p.m. Analysis of various levels along the cranio-caudal axis of the rat spinal cord revealed differences in the heavy metal content. The Timm sulfide silver staining method has demonstrated that metals in the spinal cord have a distinct regional distribution. To obtain a differentiation between the stainable metals, the effects of six chelating agents (DEDTC, dithizone, oxine, EDTA, dipyridyl, and phenantroline) on the Timm pattern were tested. EDTA left the pattern unchanged, while the other compounds showed individual differences in their influence on the Timm pattern, suggesting that the heavy metal pattern of the spinal cord consists of multiple compartments. The effect of intravital multiple low dose treatment with three of the chelating agents on the histochemical pattern and the metal content of the spinal cord was also investigated. It was found that a decrease in the metal content was not followed by reduction of stainability and vice versa.     

Heavy metals in the spinal cord of normal rats and of animals treated with chelating agents: A quantitative (Zinc,copper, and lead) and histochemical study

Summary The amounts of zinc, copper, and lead in the rat spinal cord were determined by means of flameless atomic absorption spectrophotometry. Zinc was present in a concentration about 100 p.p.m. (dry weight), copper in a concentration about 5 p.p.m., and lead in slightly more than 1 p.p.m. Analysis of various levels along the cranio-caudal axis of the rat spinal cord revealed differences in the heavy metal content.The Timm sulfide silver staining method has demonstrated that metals in the spinal cord have a distinct regional distribution. To obtain a differentiation between the stainable metals, the effects of six chelating agents (DEDTC, dithizone, oxine, EDTA, dipyridyl, and phenantroline) on the Timm pattern were tested. EDTA left the pattern unchanged, while the other compounds showed individual differences in their influence on the Timm pattern, suggesting that the heavy metal pattern of the spinal cord consists of multiple compartments.The effect of intravital multiple low dose treatment with three of the chelating agents on the histochemical pattern and the metal content of the spinal cord was also investigated. It was found that a decrease in the metal content was not followed by reduction of stainability and vice versa.     

Contribution of zinc fraction to available zinc extracted,by some chemical methods,from rice growing soils of North-Western Himalayas

A laboratory and greenhouse investigation was undertaken to study the distribution and contribution of zinc fractions to available zinc in submerged rice. Most of the total zinc was present as Al- and Fe-oxide bound (52.8%) and residual zinc (27.8%). The exchangeable (non-specifically and specifically absorbed), organically bound and Mn-oxide bound zinc fractions averaged 0.7, 1.1, 6.3 and 4.9 per cent of the total zinc, respectively. 0.1 M HCl, EDTA-(NH₄)₂CO₃ and dithizone extractants showed significant correlation with per cent yield, Zn concentration and zinc uptake by grain and the critical limits were 3.0, 1.9 and 1.0 µg^–1, respectively. Organically bound zinc exhibited significant correlation with per cent yield and zinc uptake by grain whereas specifically absorbed zinc correlated with Zn concentration in grain. Mn-oxide boudn zinc and Al- and Fe-oxide bound zinc fractions were also correlated with zinc concentration and zinc uptake by grain.     

Neo-Timm and selenium stainable glial cells of the rat telencephalon

The Neo-Timm and selenium methods predominantly stain the neuropil of the rat brain and have been found to visualize zinc in synaptic vesicles. A fraction of glial cells and neuronal somata is also stained, especially when the Neo-Timm method is used. In the present study the localization and appearance of stained glial cells in the rat telencephalon are described using the two methods and the effect of metal chelating agents on the stained glial cells is examined. Neo-Timm stained glial cells were observed in both white and grey matter, with a preponderance in the major fiber tracts of the telencephalon, and were seen to contain rather large silver grains in their cytoplasm. Chelation with diethyldithiocarbamate (DEDTC) or dithizone prevented this staining. Brains from rats treated intravitally with selenium contained only occasionally stained glial cells. However, when present they showed the same characteristics as the Neo-Timm stained glial cells, including the reaction to chelation. Although both the Neo-Timm and selenium methods primarily visualize zinc in the neuropil of the rat brain, the possibility that copper could contribute to the glial cell staining cannot be ruled out. This possibility is further discussed.     

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus. A microbeam PIXE study. Particle Induced X-Ray Emission

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus was investigated using PIXE (Particle Induced X-Ray Emission) spectroscopy. Using the proton microbeam (60 X 60 microns), 2 mm line-scans were made on hippocampal sections and the data were expressed as absolute zinc concentrations. Values of 55 and 136 ppm (dry weight) were found for the mean background zinc level and the maximum mossy fiber zinc level, respectively, in animals fed a control diet containing 50 ppm zinc. Treatment of these animals with dithizone caused about 50% reduction in the maximum mossy fiber zinc level. Feeding a zinc-deficient diet for 28 days did not cause a decrease in the mossy fiber zinc level, however, feeding the zinc-deficient diet for 90 days reduced the maximum mossy fiber zinc level by about 30%. The results are discussed in relation to the behavioral abnormalities that have been observed in zinc-deficient animals.     

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.     

Development and characterization of simulant pancreatic islets

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.     

Effects of chelating reagents on the hippocampal EEG and histochemical Timm staining pattern in a mouse brain

A series of experiments we examined in detail the effects of four chelating reagents, dithizone, DEDTC, oxine and EDTA, upon (1) Timm's staining in the hippocampal formation, (2) brain electrical activity recorded from hippocampal and cortical electrodes, (3) recovery effect with zinc acetate or zinc sulfate on abolished brain electrical activity, (4) open field activity, (5) toxicity, (6) and heart rate. Dithizone, DEDTC and oxine influenced all measures, and the degree of effect varied directory with dose. But EDTA has not any significant effect on biological system. Resulting data were summarized in Table 4.     

Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.