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The mechanism accounted for the silver staining "fast techniques" development and its histochemical meaning. II. The binding between silver and ferric ion accounted for the silver staining development.

Results obtained on filter paper strips models show that some protein and fatty acids become argentophil as an effect of a previously binding ferric ion although, in this instance, the silver staining can only be accomplished by using a silver diamine solution, since a silver nitrate solution is not effective. However, if the filter paper model is previously treated by a "multidentate ligand" before the silver nitrate solution treatment becomes able in doing the silver staining. This result shows that the binding between Fe3+ and Ag+ can be done if a suitable negatively charged "multidentate ligand" has been connected between them. The following "multidentate ligands" were tested: thiocarbohydrazide, carbohydrazide, and hexamethylenetetramine: ammonia was also tested, as an attempt to disclose if it is able to act as a "multidentate ligand". The semicarbazide effect was analysed and compared with the "ligands". It was found, either on filter paper strip models or on histological sections, that every "multidentate ligand", as well as ammonia, are effective in producing silver staining whereas semicarbazide is devoid of effect. The "ligands" effectiveness depends upon the solution pH, in the same manner that is does occur when the silver diamine "fast technique" is used on tissue sections. Histophotometric measures, taken on tissue sections, show that the silver staining afforded by the silver diamine "fast technique" is similar to that displayed by the "multidentate ligand" plus a silver nitrate solution treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Histological Techniques

[Serum and tissue silver levels after burns treated with silver compounds].

This study was performed in order to investigate serum and tissue silver levels in burns which were used 10 percent silver nitrate as a topical agent. We formed four groups of animals and pulverized 10 percent silver nitrate solution to the first group (GI) that included ten rabbits of which backs were burned by boiling water and silver sulphadiazine cream to second group (GII) with nine rabbits. We carried out 10 percent silver nitrate solution to the first control group (GIII) and silver sulphadiazine cream to the second control group (GIV) each of which had seven animals with unburned skin. We obtained blood samples from every animal before and after application of topical agent on the 1st, 3rd, 7th, 15th, 21st and 28th. We determined serum and tissue silver levels by atomic absorption spectrophotometer in kidney and liver of the animals which were sacrificed on the 28th day. In first and second groups we found that serum silver values reached on 3rd day to the maximum level and then the values decreased gradually. We also determined that diminution of the serum silver levels were prominent following on 15th day. It was shown that there was no silver in the serum on 28th day except four animals. The silver deposition in the liver was much more than in the kidney. Between these two groups there was significant difference neither in the serum on the same days nor the tissue silver levels. According to these data it was concluded that serum and tissue silver levels with 10 percent silver nitrate used in burns produced no difference from that of 1 percent silver sulphadiazine cream.

Administration, Topical

The mechanism accounted for the silver staining "fast technique" development and its histochemical meaning: I. The correlation between the silver staining intensity and the tissue bound ferric ion and some other tissue histochemical reactivities.

The mechanism accounted to accomplish the silver staining "fast technique" on tissues sections was studied towards the correlation among histophotometric measures concerning the silver staining intensity and the intensity provided by some histochemical reactions performed on spleen and liver sections from rats and pigs. By treating previously these histological sections with thioglycolate or oxalate solutions in progressive concentrations and afterwards subjecting them to a silver staining "fast technique", it was demonstrated that the silver staining intensity decreases proportionally to the thioglycolate or the oxalate solution concentration. The regression line of the silver staining intensity on the thioglycolate or the oxalate solution concentration was established, as well as its regression coefficient. On the other hand, on histological sections previously subjected to the thioglycolate or the oxalate treatment, some histochemical techniques were performed and the histophotometric measures concerning the intensity of each histochemical technique used were taken and its regression line, as well as its regression coefficient were established. By comparing the silver staining intensity regression line (or its regression coefficient) with the regression lines established for the histochemical techniques used, it was tested the correlation between the silver staining and the reactivity of some reactive groups contained into the tissues. In this manner, the influence of such reactive groups on the silver staining development was tested. The results show that there is no correlation between the silver staining intensity and the reactivity degree of the reducing groups (-SH- and carbonyl group), the 1-2-glycolic group, as well as of some protein reactive groups (phenol, imidazole, carboxylic groups). On the other hand, taking into account the respective regression lines and its regression coefficient, the comparison between the silver staining intensity and the Prussian blue reaction intensity shows a close relationship between them. This finding suggests that for the silver staining "fast technique" the tissue bound ferric ion accomplishes a very important role and can be accounted for its mechanism and its histochemical meaning.

Animals

Factors affecting the formation of metallic silver and the binding of silver ions by tissue components.

The rate of formation of metallic silver has a maximum when plotted as a function of pH. The site of this maximum on a pH scale differs noticeably for various tissue elements. By contrast, the amount of silver ions bound to the tissue is a monotonously increasing function of the pH. A temperature rise decreases the length of the induction period and increases the gradient of the ascending section of the kinetic curve representing the formation of metallic silver. It also increases the maximum amount of silver ions bound to the tissue. An increase in the concentration (activity) of the silver ions in the impregnating bath has the same effect. Chemical composition and concentration of the complexing agent, as well as "special" ions in the impregnating bath to which earlier some definitive role has been attributed in the silver staining methods, proved to be ineffective when both pH and activity of silver ions were kept constant. Illumination of the reaction was also ineffective. The kinetic curves obtained in nonaqueous but polar media (e.g., acetone) exhibited the same qualitative characteristics as those obtained in aqueous solutions. No reaction between silver ions and tissue was observed in apolar solvents.

Histocytochemistry

Argyria: clinical implications of exposure to silver nitrate and silver oxide.

This article reports the clinical findings in a work force of 30 individuals who were exposed to silver nitrate and silver oxide. Six individuals had argyria and 20 had argyrosis (deposition of silver in the eye). Measurements of blood silver levels were included as part of the examination. The results of this examination generally support the benign nature of argyria, although the question of silver causing a decrement in kidney function and night vision is not settled. Periodic slit lamp examinations as well as monitoring of silver air concentrations are necessary to assure that engineering controls are actually limiting worker exposure to silver.

Adult

Kinetics of formation of metallic silver and binding of silver ions by tissue components.

The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be divided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.

Histocytochemistry

Ultrastructural localization of silver in rat testis and organ distribution of radioactive silver in the rat.

The deposition of silver after a single intravenous injection (2 micrograms Ag g-1 body weight) was studied in the testes of Wistar rats 24 h and 1 and 2 weeks after dosing with radiolabelled 110AgNo3 (2 micrograms Ag and 1.2 kBq g-1 body weight). Also, the temporal accumulation of silver during the experimental period was monitored in the blood, testes, epididymides, kidney, liver and brain. The subcellular distribution of silver within the testes was demonstrated by using the histochemical technique of autometallography. Silver was cleared rapidly from the blood. After an initial rise, concentrations in organs remained almost stable throughout the experimental period. Silver was especially abundant in interstitial macrophages and in the basement membrane. Deposits of silver were found in all cell types of spermatogenesis and in the lysosomes of the Sertoli cells.

Animals

Serum concentrations and accumulation of silver in skin during three months treatment with an anti-smoking chewing gum containing silver acetate.

Silver acetate chewing gum was used for 12 weeks as a smoking deterrent in 21 adults. The effect of silver on serum concentrations, its accumulation in the skin and the risk of developing clinically evident argyria were investigated. Serum concentrations of silver clearly rose after chewing gum use had started, and concentrations quickly returned to normal after use had ceased. In most cases the number of silver granules in skin biopsies, observed by autometallography, increased after the gum had been used for 12 weeks. No one developed clinical signs of argyria. Silver acetate containing remedies can be used as an aid to stop smoking, but the consumption must be monitored to avoid accumulation of larger amounts of silver in the body.

Acetates

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.

Animals

Silver binding to rabbit liver metallothionein. Circular dichroism and emission study of silver-thiolate cluster formation with apometallothionein and the alpha and beta fragments.

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.

Animals

Coloration of silver-stained protein bands in polyacrylamide gels is caused by light scattering from silver grains of characteristic sizes.

This study investigates the physical basis of color effects in the detection of proteins in polyacrylamide gels by silver staining. Specifically, the hypothesis that different colors may correlate with the development of silver grains of characteristic sizes was investigated by electron microscopy. Protein bands that stained brown, yellow, and blue were excised from stained gels and prepared for electron microscopy by thin-sectioning. In each case, the size distributions of globular silver grains were determined directly from the electron micrographs. We found that blue bands have larger silver grains (with diameters of 40-100 nm) than yellow (21-39 nm) or brown bands (17-35 nm). On the basis of these and other observations, a general mechanism is proposed whereby chemical specificity of electrophoretically separated proteins is expressed in color-specific silver staining.

Electrophoresis, Polyacrylamide Gel

Comparison of silver sulfadiazine 1% with chlorhexidine digluconate 0.2% to silver sulfadiazine 1% alone in the prophylactic topical antibacterial treatment of burns.

Wound bacterial colonization in 118 patients treated with chlorhexidine digluconate 0.2% in silver sulfadiazine 1% applied daily to the burn wounds was compared to that of 135 comparable patients similarly treated with silver sulfadiazine 1%. With chlorhexidine digluconate 0.2% in silver sulfadiazine 1%, colonization by Staphylococcus aureus was less frequent (38%) than with silver sulfadiazine (54%, p = 0.016). No statistical difference was found for colonization by Enterococcus faecalis, Pseudomonas aeruginosa, or Enterobacter cloacae. Washing of the wounds of 65 patients with chlorhexidine gluconate 4% during daily dressing changes was associated with reduced wound colonization by S. aureus (35% versus 51%, p = 0.03) and P. aeruginosa (8% versus 16%, p = 0.08) when compared to the 188 washed with nonantibacterial soap. Chlorhexidine, whether added to the topical agent silver sulfadiazine (chlorhexidine digluconate 0.2%) or in the bath soap (chlorhexidine gluconate 4%), decreased colonization by S. aureus.

Administration, Topical

Sulfated glycoconjugates demonstrated in combination with high iron diamine thiocarbohydrazide-silver proteinate and silver acetate physical development.

Sulfated glycoconjugates in epithelial cells and mesenchymal cells were investigated after staining with high iron diamine-thiocarbohydrazide-silver proteinate. One purpose of the experiment was to apply a new physical developed to the staining. Instead of silver nitrate, silver lactate or silver bromide, we used silver acetate as an ion donor. This new method allowed physical development under normal lighting conditions, and resulted in the reduction of background staining even after amplification. As the developer did not contain gum arabic, troublesome treatment was not necessary. The time required for staining was very short and the electron density of the final reaction product was high and easily identifiable under the electron microscope. Fixing was not necessary. Very small amounts of reactive substance were detectable after physical development. This developmental procedure has been applied to both the preembedding staining and postembedding staining of sulfated glycoconjugates. The results obtained using this method are presented.

Acetates

A comparison between low background silver diammine and silver nitrate protein stains.

Several methods of silver staining of proteins after sodium dodecyl sulfate-electrophoresis in polyacrylamide gels were compared. The most rapid methods were found to be less sensitive than more time-consuming methods. Among the long methods, those using glutaraldehyde treatment of the gel and silver diammine complex as the silvering agent were found to be the most sensitive, at the expense of the use of a modified polyacrylamide matrix and higher silver concentrations.

Ammonia

Sensitivity to silver in a patient treated with silver sulphadiazine (Flamazine).

This report describes a 43-year-old housewife who suffered a burn and then was suspected of having a sensitivity reaction to Flamazine cream (1 per cent silver sulphadiazine). Patch testing subsequently showed she was sensitive to silver and to cetyl alcohol, but to no other constituents of Flamazine cream. The patient herself knew she was sensitive to silver but was never asked about this possibility, or told initially that Flamazine cream contained silver.

Adult

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals