Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.     

The effect of selenium on the localization of autometallographic mercury in dorsal root ganglia of rats

The autometallographic technique was used to demonstrate the localization of mercury in dorsal root ganglia of adult Wistar rats. The animals were either exposed to mercury vapour, 100 μg Hg m⁻³, 6 h day⁻¹, 5 days per week, or treated with organic mercury in the drinking water, 20 mg CH₃HgCl per litre, for 4 weeks. The effect of orally administered sodium selenite on the pattern of intracellular distribution of mercury in these two situations was investigated. In rats exposed to mercury vapour alone, faint staining was present in ganglion cells. The selenite induced a conspicuous increase in the number of stained cells and in the intracellular staining intensity. In rats treated with organic mercury, mercury deposits were detected within ganglion cells and macrophages. The number of mercury-containing cells was increased by co- administration of selenite. In addition, satellite cells, the capsule and vessel walls were faintly stained. Twenty weeks after cessation of the organic mercury treatment, mercury staining was reduced. Again, selenite treatment enhanced staining intensity. When studied using the electron microscope, mercury was restricted to lysosomes, irrespective of treatments. The present study shows that the deposition of autometallographic mercury in the dorsal root ganglia depends on the chemical type of mercury, the co-administration of selenite and the length of the survival period. This revised version was published online in November 2006 with corrections to the Cover Date.     

Autometallographic detection of mercury in rat spinal cord after treatment with organic mercury.

Autometallography was used to localize mercury in rat spinal cord after intraperitoneal administration of methylmercuric chloride (200 micrograms CH3HgCl daily). The technique permits small amounts of mercury sulfides and mercury selenides to be visualized by silver-enhancement. Mercury deposits were observed by light microscopy only in neurons. In all of the spinal cord segments selected (first cervical segment, C1; fifth cervical segment, C5; sixth thoracic segment, T6; and first lumbar segment, L1) the mercury was observed with cumulative dosages of 6000 micrograms CH3HgCl and greater. Laminae VII, VIII, and IX contained the majority of stained neurons, whereas laminae IV, V, VI, and X had a relatively lower density of mercury-containing neurons. Stained neurons were confined to specific cell groups, such as Clarke's column, nucleus intermedio-lateralis, nucleus cervicalis centralis, and nucleus dorsomedialis. At the ultrastructural level, mercury deposits were restricted to lysosomes of neurons and occasional accumulations in the lysosomes of ependymal cells.     

Enhancement of gamma-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury.

Glutathione (GSH), a major cellular antioxidant, is elevated 2- to 3-fold in kidneys of rats during prolonged treatment with mercury as methyl mercury hydroxide (MMH). Increased renal GSH is accompanied by a dose- and time-related elevation in the relative abundance of mRNA hybridizable to a cDNA probe which encodes renal gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Renal GCS mRNA is maximally elevated 4.4-fold at 3 weeks following initiation of MMH treatment. Enhancement of GSH and GCS mRNA content corresponds to a relative sparing of renal cells from oxidative tissue damage during MMH exposure. These observations suggest that increased synthesis of GSH at the genetic level occurs as an initial adaptive response to mercury-induced oxidative stress in kidney cells.     

Allosteric inhibition of rat liver and kidney arginase by copper and mercury ions

Two isozyme forms of arginase are found in the rat. All arginases are metalloenzymes which require manganese for activity. Many arginases are activated by cobalt and nickel ions and inhibited by heavy metal ions. The purpose of this study was to compare the effect of other heavy metal ions on the rat liver isozyme (arginase I) and the rat kidney isozyme (arginase II). The activation and inhibition of arginase I and II by metal ions were different. However, both isozymes were strongly inhibited by cupric and mercuric ions. The inhibition of arginase I by cupric and mercuric ions was increased greatly by preincubation of the enzyme with the metal ions. However, preincubation of arginase II by cupric and mercuric ions had little effect on the inhibition of the enzyme. Under certain conditions the kinetics of the inhibition of both arginases I and II by cupric and mercuric ions was nonlinear allosteric.     

Effect of mercury nitrate on the histochemical activity of some dehydrogenases in primary cultures of rat kidney tubular cells

Direct effect of sublethal and lethal doses of mercury nitrate on histochemical activity of G6PDH, LDH and SDH was investigated in primary cultures of rat tubular cells. Enzyme activities were studied histochemically after administration of mercury nitrate for periods extending from 5 min. to 24 hrs and also after 2,3,4 and 5 following days. The sublethal doses of mercury nitrate containing 1 microgram and 5 microgram of mercury were found to decrease histochemical activity of the studied dehydrogenases as early as 10 and 15 minutes. Their inhibitory effect was much stronger after 24 h and depended on the dose of mercury compound. The lethal doses of mercury nitrate containing 15 microgram and 20 microgram of mercury depressed the activity of dehydrogenases within 5 min. after administration. The loss of enzyme activities usually preceded the appearance of necrotic signs in the cultured cells. It was also found that the cultured cells of kidney tubules treated with sublethal doses of mercury nitrate were usually able to regain the normal level of the enzymic activity within 2-5 days.     

Interactions of mercury in rat brain

In order to study the metabolism of mercury (Hg), its affinity to metallothionein (MT), and its influence on levels of the essential metals copper and zinc in the brain tissue of rats exposed to elemental mercury (Hg^O) vapor was investigated. The major findings were:

Protective effects of selenium against mercury toxicity as studied in the rat liver and kidney by nuclear analytical techniques

Mercuric chloride and sodium selenite were separately administered to male rats in the drinking water or in a combination (2.5 mmol Hg/L and 0.1 mmol Se/L). The mercuric chloride group showed histopathological lesions, as evidenced by cell necrosis in the liver and tubular necrosis in the kidney. The sodium selenite group showed some depression in growth, but pathological changes were found neither in the liver nor in the kidney. Simultaneous administration of both compounds produced a protective effect on weight loss and histopathology. These effects were associated with some small structures in the kidney proximal tubules and to some structure in the extracellular space in the liver. Thin, unstained cryosections were freeze-dried and examined in the Studsvik Nuclear Microprobe. The structures observed in the liver and the kidney were shown to contain both selenium and mercury.     

Biochemical changes in rat kidney on exposure to elemental mercury vapor: effect on biosynthesis of metallothionein.

Evidence is presented that exposure of rats to elemental mercury vapor results in increased amounts of a metallothionein-like protein in kidney tissue but not in liver. After three or more daily exposures, each of 2 h duration, to elemental mercury vapor, more than 50% of the mercury in kidney tissue is bound to a protein having a molecular weight (mol. wt.) of about 10 000 as determined by Sephadex G-75 gel filtration chromatography. Cystine is incorporated into a 10 000 mol. wt. protein fraction from kidneys of rats which were injected with [U-14C] cystine after five daily 2-h exposures to mercury vapor. In contrast, no significant incorporation of [U-14C] cystine into this protein fraction was observed in kidneys of control rats or in livers of both control and mercury vapor-exposed rats. The in vivo incorporation of 109Cd into the fraction followed the same pattern as that of [14C] cystine in rats injected with tracer doses of CdCl2 labeled with radioactive 109Cd isotope. This 10 000 mol. wt. protein, newly synthesized in response to repeated exposures to mercury vapor, exhibited identical properties to metallothionein, namely in its subcellular localization, molecular weight, heat stability and isoelectric points. A significant incorporation of [U-14C]-cystine into this protein in rat kidney alone on exposure to mercury vapor confirms its induced biosynthesis in the kidney.     

Optical slicing and 3-D characterization of hippocampal capillaries in the rat visualized by autometallographic silver enhancement of colloidal gold particles

Summary In order to visualize the vascular system of the rat brain, 10 Wistar rats were perfused transcardially with glutaraldehyde and a 40°C gold-gelatine solution. The brains were post-fixed with glutaraldehyde and vibratomized into 100-μm-thick slices, and the gold particles were developed by autometallography. In this way, the colloidal gold particles in the vessels became encased in silver and thereby made visible. The developed gold staining is stable and does not interfere with further dehydration and counterstaining. Images were frame grabbed during optical slicing, and classic stereograms and ‘shadow’ 3-D images were produced. We found a high variation of capillary density in the hippocampal region reflecting known subregional structures. The silver-enhanced vessels acted as natural markers and made it possible to study and measure aspects of the complexity of dehydration and staining artifacts. We found a non-linear shrinking of 13–17% in the x- and y-directions and a spatial shrinking up to 50% in some regions after the dehydration and staining process. This observation may be of interest not only in relation to tissue subjected to this fixation protocol but also to other fixation procedures. The gold-gelatine autometallographic technique and the present stereograms can release data for stereological use as well This revised version was published online in November 2006 with corrections to the Cover Date.     

An autometallographic technique for myelin staining in formaldehyde-fixed tissue

A new autometallographic (AMG) technique for staining myelin in formaldehyde- or paraformaldehyde- (PFA) fixed tissue is presented. The tissue sections were exposed to AMG development without prior treatment with silver salts. The method was examined on PFA-fixed tissue from mouse, rat, pig, and formaldehyde-fixed human autopsy material. Samples from brain, spinal cord, cranial, and spinal nerves were either cut on a vibratome, frozen and cryostat sectioned, or embedded and microtome sectioned, before AMG development and counterstaining. The AMG-myelin technique results in a specific black/dark-brown staining of myelin in all parts of the CNS and PNS. It works on all species examined, independent of the histological preparation techniques applied. The AMG staining is stable, stays unchanged through decades, allows counterstaining, and has previously been used with immunohistochemical techniques. On perfusion-fixed tissue the technique works without further fixation, but the intensity of the AMG-myelin staining is increased by increased postfixation time. Additionally, immersion fixation has to last for days depending on the size of the tissue block in order to obtain proper myelin staining. The most feasible explanation of the chemical events underlying the AMG-myelin technique is that nano-sized clusters of metallic silver are formed in the myelin as a result of chemical bounds with reducing capacity, exposed or created by the formaldehyde molecule. The AMG method is simple to perform and as specific as the conventional osmium and luxol fast blue stainings. The present technique is thus an effective, simple, inexpensive, and quick myelin staining method of formaldehyde- or PFA-fixed tissue.     

Immersion autometallographic tracing of zinc ions in Alzheimer beta-amyloid plaques

An easy to perform autometallographic technique (AMG) for capturing zinc ions in Alzheimer plaques is presented. The possibility of visualizing loosely bound or free zinc ions in tissue by immersion autometallography (iZnS^AMG) is a relatively recent development. The iZnS^AMG staining is caused by zinc-sulphur nanocrystals created in 1–2 mm thick brain slices that are immersed in a 0.1% sodium sulphide, 3% glutaraldehyde phosphate buffered solution, the NeoTimm Solution (NTS), for 3 days. When the zinc-sulphur nanocrystals are subsequently silver-enhanced by autometallography, the plaques are readily identified as spheres of dark interlacing strands of different sizes, embedded in the pattern of zinc-enriched terminals. The zinc specificity of the iZnS^AMG technique was tested by immersion of brain slides in the chelator DEDTC prior to the NTS immersion. The iZnS^AMG detection of zinc ions is easily standardized and can be used in the quantification of plaques with stereological methods. This technique is the first to detect zinc in plaques in the cerebellum of transgenic PS1/APP mice and the first to detect zinc ions in plaques and dystrophic neurites at electron microscopical levels.     

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.