Uptake and distribution of chromium in Saccharomyces cerevisae exposed to Cr(III)-organic compounds

Abstract

The present study investigates the influence of different Cr(III)-organic compounds [Cr(III)-citrate and Cr(III)-histidine] in growth-nonsupportive exposure medium on the uptake and localisation of chromium in the cell structure of the yeast Saccharomyces cerevisae. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. Chromium accumulation potential was shown to depend on treatment time, metal concentration as well as the nature of the bound ligand. Chromium uptake was characterised by a time-dependent increase of total chromium which suggests that the amount of cell-accumulated chromium also tended to increase over time. Cellular chromium accumulation (mg g^?1 dry wt) of Cr(III)-histidine is higher than Cr(III)-citrate. The pH dependence pattern of chromium accumulation is similar for both of the Cr(III)-organic compounds: pH 6.5>pH 5>pH 8. Substantial differences were found between the two Cr(III)-organic compounds, in the total chromium accumulation as well as in the distribution in yeast cell walls and spheroplasts.     

The Fixing and Staining of Liriodendron Tulepifera Root Tips and their Mycorrehizal Fungus

Root tips of Liriodendron Tulipifera forming with a fungus of the Mycelium Radicis group an endotrophic mycorrhiza, were subjected to several different fixations in which the action of cationic chromium and anionic chromium were compared. Anionic chromium in the form of chromic acid was combined with several substituted benzene compounds while cationic chromium in the form of chromic sulfate (Cr₂(SO₄)₃·15 H₂O) in 4% formaldehyde (HCHO) was used with the same ring compounds. In addition, several fixatives not containing chromium were tested.

Five percent chromic sulfate (Cr₂(SO₄)₂·15 H₂O) in 4% formaldehyde (HCHO) or in 1% osmic acid with saturated aqueous salicylic and/or picric acid preserved the histological and cytological details of the mycorrhiza, as was clearly demonstrated when followed by staining in 3% acetic acid saturated with orseillin BB and counterstained with 1% crystal violet in clove oil.

Two percent ferric chloride (Fe Cl₃) in 4% formaldehyde showed the relationship of the tannin contents of the cells to the invading hyphae when followed by suitable staining.

Cationic chromium appeared to be superior to anionic chromium in preserving cell walls as well as the general histologic features of the material investigated.     

The composition and properties of Gallocyanin-chrome alum stains

Synopsis The composition of 5 common Gallocyanin-chrome alum (GCA) preparations were studied by thin layer chromatography, electrophoresis and spectrophotometry. The GCA preparations were found to be mixtures of one or more Gallocyanin-chromium co-ordination complexes, chromic ions, and, usually, free Gallocyanin. The differences in composition of the various preparations were due to differences in the preparative boiling times. The differences in histological staining properties depended on the concentrations of free Gallocyanin.The staining action of the GCA mixtures was similar to that of a typical basic dye such as Pyronin Y, both with regard to the materials stained and the effects of varying the pH and salt content of the dyebath.The chemistry of the commonest GCA co-ordination complex was investigated. It was found to have the composition 21 Gallocyanin: chromium (G₂Cr). The chromic ion was chelated to the aminocarboxylic acid. The complex carried a net positive charge in the pH range 1–9, and would thus be expected to behave similarly to basic dyes. In fact GCA was no more specific for nucleic acids than other basic dyes.     

Redox Reactions in Chromium-Containing Fixatives for Biological Materials

Biophysics - Abstract—A study of redox reaction kinetics in chromium-containing fixatives (mixtures of chromic acid, bichromate, formaldehyde, and acetic acid) showed that these fixatives...     

Collection of chromium (III) by sulfite and sisulfite chitosans

Abstract

Chromium (III) collection on sulfite (CS) and bisulfite (CBS) chitosans was investigated in order to obtain information about chromium recovery from tannery wastes from the chromium leather process. Collection of Cr(III) by sulfite and bisulfite chitosans was fast during the first 60 minutes and was affected by the pH of the solution, contact time and temperature. Chromium collected on bisulfite was easily eluted with dilute sulfuric acid solution.     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

Further Studies on the Lyo and Desmo Components of Several Hydrolytic Enzymes and Their Histochemical Significance

This report describes additional studies of the lyo and desmo components of esterase, alkaline phosphatase, acid phosphatase, leucine aminopeptidase, and β-glucuronidase. The techniques used have already been reported (7). Enzyme diffusion occurs to different degrees in different fixatives, and varies somewhat with each enzyme. Loss of enzymatic activity during fixation occurs as a result of both inactivation due to the chemical reaction of the fixative with the enzymic protein, and diffusion of the lyo component into the fixative. The amount of diffusion into formalin can be reduced by the addition of salts, sucrose, or methocel. The pH of the aqueous medium significantly influences the removal of the lyo fraction from the tissue section. A striking similarity can be noted in the proportions of each fraction of enzyme present in the kidney of the rat, dog, and man. The procedure of fixation and paraffin embedding of tissue blocks does not wholly prevent the diffusion of the lyo component from the tissue sections when they are subsequently immersed in the aqueous incubation medium.     

Mechanisms of chromation hematoxylin stains

Summary In chromation hematoxylin sequence stains of Weigert-Smith-Dietrich type an exploration is presented of the nature of chromium binding tissue end groups, of the valency of the bound chromium and of the mechanisms and conditions of its binding.At 2–4° C prolonged (2–8 weeks) mordanting in 2.5% K₂Cr₂O₇ at pH 3.5 engenders staining with acetic hematoxylin essentially limited to histochemical ethylenic sites, completely preventable by prior aqueous bromination and unaffected by sulfation or acetylation. Erythrocytes, myelin and bile casts are examples of reactive tissue elements. At 24° C and more so at 60° C there is increased reaction of muscle, cytoplasm, nuclei and other structures; the reaction is now partially blocked by acylations and is only partly prevented by bromination, indicating participation of hydroxyl groups. Deamination decreases reaction at 60° C of protein background, but not notably of myelin, erythrocytes, or bile casts. The previously reported carboxyl binding of hot chromation oxyphilia is almost inapparent when chromation is done at 3° C. Chromic acid is less selective in its action, producing some background staining even at 3° C; K₂CrO₄ engenders no acetic or neutral hematoxylin staining, even at 6.6% and 24 hour mordanting at 24° or 60° C.Chromium deposited from dichromate or chromic acid mordanting reacts with hematoxylin solutions at pH 2.5 to 7.0, that deposited from Cr III salts reacts only at pH 6–7. Mordanting with Fe III, Fe II, Cu II and Sn II salts yields hematoxylin staining respectively from pH 2.5, 4.0, 5.0 and 2.5 upward. After K₂Cr₂O₇ mordanting brief reductions and acid treatments restrict hematoxylin staining to the same neutral pH zone as that produced after Cr III mordanting, but the pH 7 staining capacity of Cr deposited from K₂Cr₂O₇ is more resistant to extraction agents than that from Cr III solutions. It is therefore concluded that the chromium deposited in dichromate mordanting is of a higher valency than Cr III and it is suggested that Cr IV is present and bound to double bond sites in ring esters in the same manner as has been formulated for Mn and Os in the attack of KMnO₄ and OsO₄ on ethylene double bonds.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health, Bethesda, Maryland. Presented in part at the Third International Congress for Histochemistry and Cytochemistry, New York 1968.     

The binding of trivalent chromium to low-molecular-weight chromium-binding substance (LMWCr) and the transfer of chromium from transferrin and chromium picolinate to LMWCr

A recent model for the role of chromium in insulin signaling requires that the oligopeptide low-molecular-weight chromium-binding substance (LMWCr) tightly bind four chromic ions before the oligopeptide obtains a conformation required for binding to the tyrosine kinase active site of the insulin receptor. To test this model, the chromium-binding constant of LMWCr was determined, and the ability of LMWCr to remove chromium from Cr2-transferrin and the nutritional supplement chromium picolinate, Cr(pic)3, was examined. These results are consistent with the model of the mode of action of LMWCr; a Hill study indicates the four chromic ions bind to apoLMWCr in a highly cooperative fashion (n =3.47) with a binding constant of 1.54x 10(21). Chromium is readily transferred from transferrin to apoLMWCr at near neutral pH. The results also suggest that reduction of the chromic center of Cr(pic)3 may be required for the supplement to release chromium; thus, release of chromium is related to a mechanism by which Cr(pic)3 may generate hydroxyl radicals in cells.     

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

Biosorption of chromium (Cr(III)/Cr(VI)) on the residual microalga Nannochloris oculata after lipid extraction for biodiesel production

In order to increase the economic feasibility of biodiesel production from microalgae, the residual biomass after biodiesel production can be utilized as biosorbent for heavy metal removal. In this study, biosorption of chromium by residual Nannochloris oculata after lipid extraction was investigated. Increased surface area of N. oculata was observed after lipid extraction. Cr(III) removal increased as the pH increased from 2 to 6, while Cr(VI) removal was highest at pH 2 and it decreased with the increase in pH. Cr(VI) was reduced to Cr(III) in the presence of biomass under acidic conditions; X-ray photoelectron spectroscopy revealed that the converted Cr(III) was bound to the biomass. Chromium removal was significantly enhanced at high chromium concentrations, which indicates that surface reactions may occur at high chromium/biomass ratios. FTIR study indicated that phosphate and carboxyl functional groups of the biomass were mainly responsible for chromium binding.     

Lysozyme antigenicity and tissue fixation.

The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues aqueous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.     

Chromium (VI)‐induced oxidative stress,apoptotic cell death and modulation of p53 tumor suppressor gene

Chromium (VI) is a widely used industrial chemical, extensively used in paints, metal finishes, steel including stainless steel manufacturing, alloy cast irons, chrome, and wood treatment. On the contrary, chromium (III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been demonstrated to exhibit a significant number of health benefits in rodents and humans. However, the cause for the hexavalent chromium to induce cytotoxicity is not entirely understood. A series of in vitro and in vivo studies have demonstrated that chromium (VI) induces an oxidative stress through enhanced production of reactive oxygen species (ROS) leading to genomic DNA damage and oxidative deterioration of lipids and proteins. A cascade of cellular events occur following chromium (VI)induced oxidative stress including enhanced production of superoxide anion and hydroxyl radicals, increased lipid peroxidation and genomic DNA fragmentation, modulation of intracellular oxidized states, activation of protein kinase C, apoptotic cell death and altered gene expression. In this paper, we have demonstrated concentration and timedependent effects of sodium dichromate (chromium (VI) or Cr (VI)) on enhanced production of superoxide anion and hydroxyl radicals, changes in intracellular oxidized states as determined by laser scanning confocal microscopy, DNA fragmentation and apoptotic cell death (by flow cytometry) in human peripheral blood mononuclear cells. These results were compared with the concentration-dependent effects of chromium (VI) on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Chromium (VI)induced enhanced production of ROS, as well as oxidative tissue and DNA damage were observed in these cells. More pronounced effect was observed on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Furthermore, we have assessed the effect of a single oral LD₅₀ dose of chromium (VI) on female C57BL/6Ntac and p53deficient C57BL/6TSG p53 mice on enhanced production of superoxide anion, lipid peroxidation and DNA fragmentation in the hepatic and brain tissues. Chromium (VI)induced more pronounced oxidative damage in p53 deficient mice. This in vivo study highlighted that apoptotic regulatory protein p53 may play a major role in chromium (VI)induced oxidative stress and toxicity. Taken together, oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptotic regulatory gene p53 are involved in chromium (VI)induced toxicity and carcinogenesis.     

The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters

Synopsis The influence of several fixation and dehydration procedures on the retention of free cholesterol and cholesterol esters was studied in filter paper preparations. The retention of free cholesterol by the filter paper proved to be decreased by the addition of digitonin to the aldehyde fixative (aqueous phase) and was only slightly enhanced by partial dehydration (alcoholic phase, up to 70% ethanol). Furthermore, digitonin or the presumably formed cholesterol-digitonide complex bound hardly any osmium oxides in glass-fibre paper.Up to 26% of the cholesterol esters was mobilized during the aqueous phase when digitonin was added to the aldehyde fixative. When the glass-fibre papers containing the digitonin cholesterol-ester-osmate complexes were stored in distilled water after fixation, the fluid became turbid. Particulate material isolated from this turbid solution showed ultrastructurally a close resemblance to the whorls observed by several authors in tissue fixed by a digitonin-containing aldehyde fixative.Digitonin also changed the ultrastructural appearance of liposomes, containing lecithin: cholesterol: phosphatidic acid, in a molar ratio 721. Our observations lead to the conclusion that the use of digitonin-containing fixatives should be abandoned, because they give results which cannot be interpreted. By the use of K₄[Fe(CN)₆] containing OsO₄ in the post-fixation step we were able to demonstrate an increase in the visualization of membranous structures (liposomes).     

Hot chromation oxyphilia

Summary On exposure of formol or methanol chloroform fixed sections to solutions of 2.5–5% potassium dichromate or 4.2% chromic acetate for 18 hours at 60° C a strong relative oxyphilia develops. As measured with buffered solutions of azure A eosin B the point at which various tissue elements pass from red to blue staining is elevated by 3 to 4 pH units. A similar change occurs in K₂Cr₂O₇ in about 21 days at 24° C and at 4° C only a slight elevation of protein pK appears in 6 weeks. Ferric chloride and ferrous sulfate solutions of comparable metal content produce a less marked shift (about 2 pH units) and relatively slight similar action is produced by aluminium and copper salts (about 1 pH unit). Heating in water, NaCl, KH₂PO₄ and K₂CrO₄ solutions is without effect. The effect is similar to that of methylation on phosphoric and carboxylic acid sites, and combination of the two procedures is not additive. Mast cell and cartilage staining are not significantly affected. Deoxyribonucleic acid is not extracted; the Feulgen reaction persists, though 1 hour hydrolysis is required vs 12–15 minutes on control formol material. Metal binding as revealed by staining in acetic hematoxylin remains demonstrable after 10 days heating in 2.5% K₂Cr₂O₇, but disappears in 6–16 hours in chromic, ferric and ferrous salt solutions. Chemical analysis reveals the continued presence of chromium at 4–5 % dry weight levels in liver and brain, alike after chromic acetate and potassium dichromate (24 hours, 60° C), though no histochemical Cr demonstration is obtained after Cr acetate. It is concluded that carboxyl and phosphoryl residues in tissue bind chromic (and iron) ions, which are then converted by heating to an unreactive insoluble form. Removal of demonstrable chromium with acid alcohol, sodium dithionite or ß-mercaptoethanol from dichromate treated tissues does not reverse the oxyphilia.Supported by National Cancer Institute Grant No. C4816, National Institutes of Health.     

Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes

Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1-P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 microM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at 120 microM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-L-glutamate do not nick DNA to a significant degree.     

The effect of different fixatives on chromatin: Cytochemical and ultrastructural approaches

Summary This study explores the effects of two types of fixative on chromatin. The first type (acrolein, glutaraldehyde) engenders a high degree of ultrastructural preservation. The other type are fixatives that are widely used in cytochemistry and cytogenetics (acetic acid, 31 by vol. methanol-acetic acid, methanol alone, formaldehyde).Lymphocytes of adult rats so-fixedin vitro were prepared for electron microscopy or microdensitometric evaluations of smears. Assessments were made of variations in their total protein, nuclear basic protein and DNA contents. DNA was determined both as Feulgen-positive material and by its binding of intercalating dyes (Methyl Green, specific for double-stranded polynucleotides).Our results showed that some fixatives break up the chromatin organization by acting on particular components of chromatin fibres. They can thus be considered to be destructive agentsin situ. In addition, a revaluation of some aldehyde fixatives is proposed for both ultrastructural and cytochemical research.     

The effects of hydraulic retention time (HRT) on chromium(VI) reduction using autotrophic cultivation of Chlorella vulgaris

Bioprocess and Biosystems Engineering - Chromium is an acutely toxic heavy metal that is known to be a carcinogen. Of the two predominant forms of chromium, Cr(III) and Cr(VI), Cr(III) has only...     

Improved immunohistochemical localization of tissue antigens using modified methacarn fixation

Detailed comparative studies have been carried out using a wide range of aqueous and nonaqueous based fixatives in order to select the optimal fixation schedule for the immunohistochemical localization of four antigens: type IV collagen, actin, Factor VIII, and epithelial membrane antigen (EMA). Modified methacarn fixation provides an ideal combination of maximum staining and morphological preservation for these antigens. Enhanced immunoreactivity with this fixative was also noted for a broad spectrum of antigens, including myoglobin, glial fiber acidic protein, keratin, myosin, laminin, prostatic acid phosphatase, alpha-fetoprotein, and carcinoembryonic antigen. Although it is not recommended for most cell surface antigens, this fixative may have a definite place in diagnostic surgical pathology.