The ultrastructural localisation of cadmium.

A modified technique for the ultrastructural localisation of heavy metals is described in this paper. The method involves precipitation of heavy metals as sulphides in the tissue by using (NH4)2 S after brief fixation in glutaraldehyde. The sulphides are, in the presence of a physical developer, then used to catalyse the reduction of silver ions into visible molecular silver. This latter step of physical development has been normally carried out after embedding and sectioning. However, when we followed this method we found that the dark metal sulphide was lost from the tissue during the embedding in epoxy resin. Hence the method was unsuitable for our proposed experiment on the ultrastructural localisation of cadmium. We subsequently modified the technique primarily by treating very thin tissue slices with the developer before dehydration and embedding, thus eliminating any problem from sulphide loss. This modified technique was used to investigate the ultrastructural localisation of cadmium in the kidneys of mice which had been exposed to 50 ppm cadmium in their drinking water for up to eight months. The molecular silver was found to be located mainly in the proximal tubule cells, either as dense clumps in apical vesicles and lysosomes or diffuse grains throughout the cytoplasm of the cells particularly in the basal region. We interpret these results as indicating that cadmium is found in the apical vesicles, lysosomes and cytoplasm of proximal tubule cells.     

The ultrastructural localisation of cadmium

Summary A modified technique for the ultrastructural localisation of heavy metals is described in this paper. The method involves precipitation of heavy metals as sulphides in the tissue by using (NH₄)₂S after brief fixation in glutaraldehyde. The sulphides are, in the presence of a physical developer, then used to catalyse the reduction of silver ions into visible molecular silver. This latter step of physical development has been normally carried out after embedding and sectioning. However, when we followed this method we found that the dark metal sulphide was lost from the tissue during the embedding in epoxy resin. Hence the method was unsuitable for our porposed experiment on the ultrastructural localisation of cadmium. We subsequently modified the technique primarily by treating very thin tissue slices with the developer before dehydration and embedding, thus eliminating any problem from sulphide loss. This modified technique was used to investigate the ultrastructural localisation of cadmium in the kidneys of mice which had been exposed to 50 ppm cadmium in their drinking water for up to eight months. The molecular silver was found to be located mainly in the proximal tubule cells, either as dense clumps in apical vesicles and lysosomes or diffuse grains throughout the cytoplasm of the cells particularly in the basal region. We interpret these results as indicating that cadmium is found in the apical vesicles, lysosomes and cytoplasm of proximal tubule cells.     

Autometallography

Summary The autometallographic procedure represents a new technique that can substitute for the normal methods of physical development (PD). The physical developer (a solution of reducing substance, silver salt and protection colloid) is replaced by a photographic emulsion and chemical developer. Accumulations of gold, silver, metal sulphides and metal selenides can be amplified by the present technique.Tissue sections placed on glass slides are covered by a silver bromide containing emulsion, dried and exposed to a chemical developer. After development the emulsion is either removed or cleared and the sections are counterstained and embedded.The autometallographic procedure can also be applied to ultrathin sections.     

Autometallography: tissue metals demonstrated by a silver enhancement kit

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.     

Bioleaching of metallic sulphides withThiobacillus ferrooxidans in the absence of iron (II)

Bioleaching of metallic sulphides withThiobacillus ferrooxidans in the absence of iron (II) was studied using pure sulphides and mixtures. The direct mode of bacterial action was analysed with respect to sulphide solubility, exposed solid surfaces and bacterial attachment to the solids. Bioleaching of mixed sulphides showed enhancement of metal extraction in comparison with pure sulphides which suggests metal extractions would be better from polymetallic sulphide ores than from similar matrices with only one sulphide.     

Histochemical differentiation of autometallographically traceable metals (Au, Ag, Hg, Bi, Zn): protocols for chemical removal of separate autometallographic metal clusters in Epon sections

Nano-sized clusters of gold atoms, or alternatively silver, mercury, bismuth, or zinc sulphide/selenide molecules, can be autometallographically silver-enhanced by being placed in a developer containing reducing molecules and silver ions, i.e. an autometallographic developer. A specific recipe has been worked out for each autometallographically traceable metal, and in cases where two or more autometallographic catalysts are present in the same section it is feasible to distinguish one from the other by chemical removal of one or the other of the metals. In the present study we present protocols that allow differentiation and control of specificity of the established autometallographically detectable metals. It is recommended to implement a multi-element analysis, e.g. proton-induced X-ray emission on a few samples to secure the histochemical data.     

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.     

The autometallographic zinc-sulphide method. A new approach involvingin vivo creation of nanometer-sized zinc sulphide crystal lattices in zinc-enriched synaptic and secretory vesicles

Summary A new version of Timm's sulphide silver method involvingin vivo binding of zinc ions in zinc enriched terminals is presented. By injecting sodium sulphide into the vena cava of deeply anaesthetized animals, it is possible to bind chemically the vesicular zinc, i. e. chelatable zinc (zinc ions), in secretory and synaptic vesicles, in the form of zinc sulphide crystal lattices. Four minutes after the intravenous injection the animal is perfused transcardially with a phosphate-buffered solution of glutaraldehyde, glutaraldehyde and formaldehyde, or with a saline solution. The nanometer-sized catalytic crystals can then be silver-amplified in cryostat and vibratome sections by exposure to an autometallographic developer. It is demonstrated that contemporaneously with silver enhancement, the zinc sulphide crystals are transformed to the corresponding silver sulphide crystals. For ultrastructural studies, autometallographic development of vibratome sections is recommended. From these sections tissue blocks are cut from the areas of interest, blockstained with osmium tetroxide and embedded in Epon. This approach results in a zinc-specific autometallographic staining of the sections of a hitherto unseen, high technical quality.     

Dissolved and particulate trace metal geochemistry in the scheldt estuary,S. W. Netherlands (water column and sediments)

The geochemistry of dissolved and particulate trace metals has been studied in the water column and the sediments of the Scheldt estuary between 1987 and 1990. A strong seasonal influence on the behaviour of dissolved Cd, Cu and Zn is observed, related to the redox conditions in the upper estuary and phytoplankton activity in the lower estuary (which are both seasonally dependent variables). The dissolved trace metal concentrations in the fresh water end-member are remarkably low during spring and summer, due to metal sulphide precipitation in the anoxic Scheldt river. However, the dissolved concentrations increase rapidly with increasing salinity, due to oxidation of metal sulphides that are present in the suspended matter, accompanied by (e.g. chloro-)complexation of the released metals. Readsorption of Cd and Zn occurs in the lower estuary during the spring phytoplankton bloom. During winter, when the Scheldt river is not completely anoxic, much higher dissolved trace metal concentrations are observed in the fresh water end-member since metal sulphide precipitation in the water column is precluded. Rapid trace metal removal is observed in the low salinity, high turbidity zone, due to adsorption onto suspended matter and freshly precipitated iron and manganese oxyhydroxides. Upon further mixing, desorption is apparent, due to a similar oxidation-complexation mechanism as observed during spring and summer. Pore water infusion may also contribute to the enrichment of dissolved Cd, Cu and Zn in the mid-estuarine region. The trace metal contents of the suspended matter and the sediments show a continuous decrease with increasing salinity. This behaviour is to a very large extent due to physical mixing of contaminated fluvial particulates and relatively unpolluted marine particulates. Desorption of Cd, Cu and Zn can be identified but is of minor importance compared to the conservative mixing process. The distribution of dissolved Cd, Cu and Zn in the pore waters of the mid-estuarine region reflects the impact of early diagenetic processes. Trace metal peaks are observed near the sediment-water interface, and at greater depth in the manganese and iron reduction zones. These peaks are attributed to oxidation of reduced trace metal compounds (e.g. sulphides) and reduction of the (iron and manganese) oxide carrier phases, respectively. At greater depth, the dissolved trace metal concentrations are much lower due to metal sulphide precipitation in the sulphate reduction zone. Analysis of a large sediment dataset indicates severe trace metal pollution of the Scheldt estuary at the end of the fifties. A major reduction of the pollution by As, Cr, Hg, Pb, and Zn has occurred in the seventies, and of Cd and Cu in the eighties. The Ni pollution has increased over the time period considered. In spite of this improvement, the present-day pollution status of the Scheldt estuary is still reason for concern.     

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Metal-catalyzed oxidation renders silver intensification selective. Applications for the histochemistry of diaminobenzidine and neurofibrillary changes

Physical developers can increase the visibility of end products of certain histochemical reactions, such as oxidative polymerization of diaminobenzidine and selective binding of complex silver iodide ions to Alzheimer's neurofibrillary changes. Unfortunately, this intensification by silver coating is generally superimposed on a nonspecific staining originating from the argyrophil III reaction, which also takes place when tissue sections are treated with physical developers. The present study reveals that the argyrophil III reaction can be suppressed when tissue sections are treated with certain metal ions and hydrogen peroxide before they are transferred to the physical developer. The selective intensification of Alzheimer's neurofibrillary changes requires a pre-treatment with lanthanum nitrate (10 mM/liter) and 3% hydrogen peroxide for 1 hr. The diaminobenzidine reaction can be selectively intensified when physical development is preceded by consecutive treatments with copper sulfate (10 mM/liter, pH 5, 10 min) and hydrogen peroxide (3%, pH 7, 10 min). In peroxidase histochemistry, this high-grade intensification may help to increase specificity and reduce the threshold of detectability in tracing neurons with horseradish peroxidase or in immunohistochemistry when the peroxidase-antiperoxidase method is used.     

Engineering the active site of ascorbate peroxidase.

The oxidation of a number of thioethers, namely methyl phenyl sulphide (1), ethyl phenyl sulphide (2), isopropyl phenyl sulphide (3), n-propyl phenyl sulphide (4), p-chlorophenyl methyl sulphide (5), p-nitrophenyl methyl sulphide (6) and methyl naphthalene sulphide (7), by recombinant pea cytosolic ascorbate peroxidase (rAPX) and a site-directed variant of rAPX in which the distal tryptophan 41 residue has been replaced with an alanine (W41A) has been examined. The electronic spectrum (pH 7.0, mu = 0.10 M, 25.0 degrees C) for the ferric derivative of W41A (lambda(max)/nm = 411, 534, 560, 632) is indicative of an increased quantity of 6-coordinate, high-spin and/or 6-coordinate, low-spin haem compared to rAPX. Steady state oxidation of sulphides 1-4 and 7, gave values for kcat that are approximately 10-fold and 100-fold, respectively, higher for W41A than for rAPX. For rAPX, essentially racemic mixtures of R- and S-sulphoxides were obtained for all sulphides. With the exception of sulphide 7, the W41A variant shows substantial enhancements in enantioselectivity, with R : S ratios varying between R : S = 63 : 37 (sulphides 1 and 4) and R : S = 85 : 15 (sulphide 6). Incubation of sulphide 2 with rAPX or W41A and [(18)O] H(2)O(2) shows 95% (rAPX) and 96% (W41A) transfer of labelled oxygen to the substrate. Structure-based modelling techniques have provided a fully quantitative rationalization of all the experimentally determined R : S ratios and have indicated that reorientation of the sidechain of Arg38, such that access to the haem is much less restricted, is influential in controlling the stereoselectivity for both rAPX and W41A.     

Light and electron microscopic localization of silver in biological tissue

Summary A method is described that visualizes trace amounts of silver in frozen, paraffin and epon sections from biological tissue. After exposure to light, which ensures reduction of silver ions that are not bound to sulphide, histological sections from animals treated with silver compounds are exposed to a photographic developer containing silver ions. Tissue silver acts as a catalyst for the hydroquinone reduction of silver ions to metallic silver which then accumulates at the site of the trace deposit. Light and electron micrographs showing silver in different organs from albino rats treated with silver lactate are presented. Localization of silver in motor neurons of the spinal gray matter and pons indicates a transport of silver over the blood-brain barrier. Silver precipitates in fetal liver suggest that silver ions can penetrate the placental barrier.     

A rapid screening method for the isolation of metal-accumulating microorganisms

Summary An agar plate screening method was developed for the rapid isolation of heavy metal-accumulating microorganisms and preliminary estimation of their biosorption capacity. The test is based on the visulaization and interpretation of the metal distribution between agar and colonies by chemical preciptitation with hydrogen sulphide or ammonium sulphide. The heavy metals silver, thallium, lead, copper, nickel and cadmium have been tested successfully. The efficiency of the method is demonstrated for isolating silver-accumulating bacterian and estimating silver biosorption capacity.     

The reaction of zinc sulphide with ionic copper—the biological implications. Part 1. Surface area studies of zinc sulphide and its reaction with copper complexes and copper containing proteins

The role of metal sulphides vis-à-vis the availability of dietary copper in ruminant animals has been investigated using zinc sulphide as a model metal sulphide and a selection of copper complexes and copper containing proteins as models for sources of dietary copper. The extent of reactivity of zinc sulphide towards the copper complexes is dependent upon the type of donor atom co-ordinated to copper:

The order to reactivity is found to be CuO > CuN > CuS complexes and is in keeping with the reported values for the instability constant pK_n of the complexes. In contrast, no reaction is observed between zinc sulphide and the copper containing proteins studied (azurin, superoxide dismutase and cerulophasmin) and is attributed to the protection of the copper centres by the protein backbone. The results facilitate an understanding of copper metabolism in ruminants and a mechanism is proposed for the removal of dietary copper sources in such species.Reactions between copper(II) sulphate solutions and samples of zinc sulphide having a range of specific surface areas (prepared by sintering at differing temperatures) have been studied. The fact that the reactivity is found to be highly dependent upon the specific surface area of the metal sulphide may well be of significance when considering the fate of copper in sulphur-rich biological systems.     

Application on Timm sulphide silver method for electron microscope localization of lead ions in blood cells.

The application of the Timm sulphide silver method for the demonstration of lead ions in peripheral blood cells is investigated, using male white adult Wistar rats treated with a single dose of lead acetate, range 150 mg/kg b.w. To improve the Timm reaction for electron microscopy, fixation of whole cells with glutaraldehyde fixative solution saturated with H2S, an agarose embedding and physical development of thick sections without prior cryostat sectioning are presented. At 6.0 hours after injection both erythrocytes as well as white cells reveal the positive Timm reaction. All types of blood cells contain numerous cytoplasmic precipitates illustrating the intracellular lead accumulation. It is shown that the invaginations of white cell nuclear membrane possess a storing function as areas of lead depots. As a rule, the neutrophils display a highest amount of cytoplasmic precipitates and exclusively a low amount of reaction products in basophils is observed. At 14 days after injection, precipitates are present only in erythrocytes and monocytes. A suggestion of a possible functional significance of changes in the Timm staining pattern in blood cell types is discussed in this paper.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Metal species indicate the potential of constructed wetlands for long-term treatment of metal mine drainage

Experimental peat-based wetlands at the former Bell Copper Mine (Smithers, B.C., Canada) removed copper from low strength (0.3–1.0 ppm, pH 6–8) and high strength (35–50 ppm, pH 3) copper-contaminated mine drainage. Copper species retained in wetlands sediments were quantified as a means to assess the long-term potential of these wetlands for treating mine drainage. Previous microbial and chemical analyses had indicated that sulphide was biologically generated in these wetlands, and mineralogical analysis confirmed that copper sulphides were present in wetland sediments. However, sequential leaching of these sediments showed that copper was predominantly in organically-bound and oxide-bound phases, with a smaller proportion present as sulphides. The available data suggest that much of the copper was retained as sulphides when low strength mine drainage was applied to the wetlands.