Depression of bone marrow colony formation in gold-induced neutropenia.

Bone marrow culture in semi-solid agar was used to assess the proliferative activity and the response to sodium aurothiomalate of the myeloid precursor cells from patients during and after recovery from neutropenia associated with the use of this drug. Colony formation was reduced during the neutropenia and returned to normal after recovery. The rheumatoid process itself did not impair colony formation even in patients with Felty''s syndrome. Sodium aurothiomalate inhibited colony formation by normal marrow in a dose-dependent manner. Bone marrow colonies from patients who had recovered from neutropenia induced by sodium aurothiomalate were not abnormally sensitive to the inhibitory effect of the drug in vitro. The metabolism of gold is probably altered in a small proportion of patients, which causes high local concentrations within the bone marrow leading directly to marrow depression.     

Ploidity and cell cycle progression during treatment with gold chloride, auranofin and sodium aurothiomalate. Studies on a human epithelial cell line and its sub-strains made resistant to the antiproliferative effects of these gold compounds

The possible influence of gold(III) chloride and the two gold(I)-containing anti-arthritic drugs, auranofin and sodium aurothiomalate, on cellular ploidity and cell cycle progression was investigated on cultured human epithelial cells. Four different cell lines were used: the parent line (HE) and three sub-strains which previously had acquired resistance to the antiproliferative effects of either 350 mumol gold chloride/l culture medium (HEAu350), 2 mumol auranofin/l (HEAF) or 300 mumol sodium aurothiomalate/l (HEMyo). DNA-histograms were obtained by flow cytometry examinations during a 9-days' exposure to either of these gold-containing compounds and concentrations. The HE, HEAF and HEMyo cells had similar ploidities, close to tetraploid. The HEAu350 cells had altered ploidity to distinct tetraploid. The distribution of the resistant cells with the cell cycle phases was not different from that of untreated HE cells. The HE cells, when treated with auranofin or sodium aurothiomalate, accumulated in the G2-phase of the cell cycle. In addition, a new cedecoploid peak appeared. No such changes were observed on gold chloride exposure or in HE controls grown without drug supplement. The effects of auranofin and sodium aurothiomalate on cell cycle progression of the HE cells possibly indicate a tendency to polyploidity, and furthermore that inhibition of cellular mitosis is one mechanism of the antiproliferative effect common to the two drugs.     

Characterization of protein-bound gold in rat urine following aurothiomalate administration and of rat and human albumin-gold-thiomalate

The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed.     

Covalent labelling of histones with aurothiomalate. Gold-labelled H2A-H2B dimers assemble to octamers, which form crystalline helical tubes

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.     

Effects of gold(I) antiarthritic drugs and related compounds on Pseudomonas putida.

The effects of the antiarthritic drugs aurothiomalate (AuTm), aurothioglucose (AuTg), auranofin, its metabolite triethylphosphinegold(I)thioglucose (Et3PAuTg), and several related complexes on the growth of Pseudomonas putida were studied. Two strains were used, one of which (BK135) was more sensitive to Et3PAuTg (tolerant up to 4 microM) than the other (BK403; tolerant to at least 500 microM). Gold thiolate complexes and thiolate ligands alone had little effect on growth. Gold phosphine complexes increased the length of the lag phase of growth and reduced oxygen uptake. Marked changes in cellular morphology were determined by electron microscopy. Copper(II) compounds and aurothiomalate were synergistic in their growth inhibitory effects towards these bacteria. Experiments with 195Au suggested that a mechanism does not exist for the short term (minutes) uptake of gold by sensitive or resistant bacteria, but the resistant strain appeared to limit gold uptake over a longer term (hours).     

Evidence that suramin and aurothiomalate are teratogenic in rat by disturbing yolk sac-mediated embryonic protein nutrition

Suramin (250 mg/kg) and sodium aurothiomalate (100 mg/kg) both induced congenital malformations in the offspring following treatment of pregnant rats at either 8.5 or 9.5 days of gestation. Conceptuses from 9.5-day pregnant rats were cultured for 48 h in homologous serum to which either suramin or sodium aurothiomalate was added for the final 6 h. The presence of suramin up to 5 mg/ml had no effect on the protein content of yolk sacs at harvesting, but at 10 mg/ml caused a significant decrease. In contrast sodium aurothiomalate increased the protein content of yolk sacs at harvesting, in a concentration-dependent manner up to 100 micrograms/ml. Neither suramin nor sodium aurothiomalate significantly affected embryo protein content. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. When suramin (2-10 mg/ml) was present for the final 6 h of culture, the quantity of radioactivity measured in the yolk sac at harvesting was significantly decreased in a concentration-dependent manner. No radioactivity was detected in the embryos. Sodium aurothiomalate had no effect on the uptake of 125I-labelled polyvinylpyrrolidone. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in the conceptus (both yolk sac and embryo) at harvesting. Suramin (5 mg/ml), present for the final or penultimate 6 h, significantly decreased the uptake of radioactivity into conceptuses and caused a significant increase in the proportion of the captured radiolabel that was associated with the yolk sac. Sodium aurothiomalate (25 or 500 micrograms/ml) had no effect on the total uptake of radio-label but caused a significant increase in the proportion of total radioactivity captured that was associated with the yolk sac. These data indicate that suramin, by interfering with both the uptake and intralysosomal digestion of protein, and sodium aurothiomalate, by inhibiting digestion of captured protein, disturb the normal pathway of yolk sac-mediated protein utilization with a consequent diminution of the supply of amino acids to the conceptus. The effects of suramin are seen only at high concentration, those of sodium aurothiomalate at much lower concentrations. It is likely that the two drugs exert their teratogenic action by their effects on the yolk sac nutritional pathway with resultant amino acid deprivation of the conceptus at a critical stage of development.     

Sulphasalazine in rheumatoid arthritis: a double blind comparison of sulphasalazine with placebo and sodium aurothiomalate.

Uncontrolled studies have suggested that sulphasalazine may be an effective second line agent in rheumatoid arthritis. Sulphasalazine was therefore compared with placebo and intramuscular sodium aurothiomalate in 90 patients with active rheumatoid arthritis. After six months'' treatment both sulphasalazine and sodium aurothiomalate had produced significant clinical and laboratory benefit, whereas placebo had produced no significant change in any variable. Thirteen patients stopped taking the placebo because of lack of effect whereas only two patients stopped taking sulphasalazine and one sodium aurothiomalate for this reason. The major toxicity encountered in the group treated with sulphasalazine was nausea or vomiting, or both; this may be related to slow acetylator phenotype. Sulphasalazine appears to be an effective second line agent, and further pharmacokinetic studies might prove useful in diminishing gastrointestinal side effects.     

Autometallographic demonstration of gold in the adrenal gland of rats exposed to sodium aurothiomalate

The presence of gold was investigated in sections of the adrenal glands from rats which had been exposed to intraperitoneal sodium aurothiomalate (32 to 120 mg). Gold was histochemically detected in cortical endocrine cells, chromaffin cells and in fibroblasts and macrophages of both the cortex and medulla. Invisible traces of gold were silver enhanced by autometallography making them readily visible at both the light and electron microscopic levels. The intracellular staining intensity was dose-dependent. In general, the number as well as the staining intensity of individual cells, were highest in the zona glomerulosa and zona reticularis. In gold-containing cells the silver-amplified deposits were present in lysosomes.     

Aurothiomalate as Preventive and Chain‐Breaking Antioxidant in Radical Degradation of High‐Molar‐Mass Hyaluronan

The potential anti‐ or pro‐oxidative effects of a disease‐modifying antirheumatic drug, aurothiomalate, to protect high‐molar‐mass hyaluronan against radical degradation were investigated along with L ‐glutathione – tested in similar functions. Hyaluronan degradation was induced by the oxidative system Cu^II plus ascorbate known as the Weissberger's oxidative system. The time‐ and dose‐dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical‐scavenging capacity based on a decolorization 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L ‐glutathione showed excellent scavenging of ^.OH and peroxyl‐type radicals, however, at the lowest concentration applied, its pro‐oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L ‐glutathione, however, at the lowest concentration tested, no significant pro‐oxidant effect was observed.     

DNA hybridization sensor based on aurothiomalate electroactive label on glassy carbon electrodes

In this work, a gold complex is used as electroactive label for monitoring hybridization assays on glassy carbon electrodes. Ionic gold is bound to a 30-mer sequence of the SARS (severe acute respiratory syndrome) virus, responsible for the atypical pneumonia, using sodium aurothiomalate. In order to label this single strand, a mixture of sodium aurothiomalate and the strand is prepared. Then, it is incubated for 24 h at 37 degrees C and, finally, free gold is separated from the labeled strand by a dialysis against a 0.15M NaCl solution (pH 7.5). The DNA hybridization sensor is designed immobilizing the complementary probe on the pre-treated electrode surface and, then, the hybridization reaction takes place with the gold labeled strand. The electrochemical determination is based on the catalytic effect of electrodeposited gold on the reduction of silver ions. In non-stringent experimental conditions, a limit of detection of 15 fmol (30 microL) is obtained, and discrimination between a complementary oligonucleotide and a three-based mismatch complementary oligonucleotide is achieved. For the discrimination of a single-base mismatch, is needed to use stringent conditions (50% of formamide in the hybridization buffer).     

The effect of second-line antirheumatic drugs on interleukin-8 mRNA synthesis and protein secretion in human endothelial cells.

Interactions between interleukin 8 (IL-8) and endothelial cells play an important role in the emigration of mononuclear cells from the blood into areas of inflammation. We examined the ability of specific second-line antirheumatic drugs to regulate (IL-8) gene expression and protein secretion in interleukin 1 (IL-1) stimulated human umbilical vein endothelial cells and peripheral blood mononuclear cells. The drugs sodium aurothiomalate, D-penicillamine and sulphasalazine were all able to modulate IL-8 mRNA synthesis in and protein secretion from endothelial cells. A bimodal effect was observed: at low concentrations IL-8 was suppressed, whereas higher concentrations resulted in an increased IL-8 production. In endothelial cells, treatment with hydrocortisone led to a linear suppression of IL-8 production in concentrations ranging from 0.5 micrograms/ml up to 500 micrograms/ml. Sulphapyridine, auranofin, hydroxychloroquine and methotrexate, had no effect on IL-8 secretion in endothelial cells. By contrast, 5-aminosalicylic acid induced a threefold increase in the IL-8 release. In peripheral blood mononuclear cells it was only possible to suppress the IL-8 production by hydrocortisone treatment. These results indicate that suppression of IL-8 production in endothelial cells could be an important factor in the mode of action for a number of second-line antirheumatic drugs.     

Caproyl esterase from rat testis: Purification and action on cumulus cells

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein. The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate. The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.     

Histochemical evaluation of sodium aurothiomalate inhibition of mouse sperm enzymes

Histochemical procedures for the mouse sperm enzymes hyaluronidase, esterase and acrosin were used to test the inhibitory effects of the low molecular weight hyaluronidase inhibitor sodium aurothiomalate (Myocrisin): hyaluronidase and esterase, but not acrosin, were inhibited. These enzymes were also inhibited in testis homogenates when assayed spectrophotometrically. These results suggest that the antifertility effects of sodium aurothiomalate may be due to the inhibition of several sperm enzymes including both hyaluronidase and esterase. These histochemical assays may be useful for in-vivo detection of chemicals that affect male fertility.     

Aurosomes produced in the synovial membrane by the oral administration of a gold compound SK & F 36914.

Chlorotriethylphosphine gold (SK & F 36914) administered orally to rabbits produced aurosomes in the synovial membrane. These aurosomes were similar to aurosomes produced by parenteral or intra-articular injections of soluble gold salts such as sodium aurothiomalate and aurothioglucose. This study shows, (1) that an orally administered gold compound is capable of producing gold deposits in the synovial membrane, and (2) that such compounds may have a future in the treatment of rheumatoid arthritis.     

Tartrate-resistant acid phosphatase: a potential target for therapeutic gold

Gold compounds are disease-modifying agents for the treatment of rheumatoid arthritis. They act on the immune system but the mechanism is not fully understood. Gold has been shown to affect antigen processing by T-cells and also reduces expression of cytokines in macrophages. Tartrate-resistant acid phosphatase (TRAP), expressed by osteoclasts, macrophages and dendritic cells is an enzyme with roles in skeletal metabolism and the immune response. TRAP is able to degrade skeletal phosphoproteins including osteopontin, identical to the T-cell cytokine, Eta-1; we thus propose that TRAP regulates the Eta-1 pathway common to the immune system and skeleton. We compared the distribution of osteopontin and TRAP in sections of 18-day-old embryonic mice by immunohistochemistry. Both proteins occurred in the same locations. To determine whether gold compounds exert their effects by modification of TRAP activity, we examined the action of gold chloride and the prodrugs, aurothioglucose and aurothiomalate on the dephosphorylation of osteopontin by TRAP. Aurothioglucose and aurothiomalate had little effect on phosphatase activity; gold chloride was a potent non-competitive inhibitor (Ki < 47 x 10(-9) M). These findings indicate a possible molecular mechanism for the action of therapeutic gold and further implicate TRAP in the control of immunity.     

A 1H nmr study of the interaction of aurothiomalate ("Myocrisin") with human red blood cells in vitro

The results of 1H spin-echo Fourier transform (SEFT) nuclear magnetic resonance (nmr) experiments suggest that some aurothiomalate binds intracellular glutathione (GSH) when added to suspensions of red cells in vitro. When added to red cell lysates, a specific binding of gold to cysteine of GSH is observed together with release of thiomalate. Gold binding to GSH can be reversed by addition of dimercaptopropanol sulfonate. Spectra are compared to those of an aurothiomalate-GSH model system. The relationship of these findings to the mechanism of action of Myocrisin is discussed.     

Photosnesitization of DNA by gold.

Au (III) reacts with DNA at pH 5.6 to form a complex which is sensitive to mid-UV radiation. Cyclobutane pyrimidine dimers are produced at some 15 to 30 times the rate that they are in untreated DNA. The mechanism of photosensitization appears to involve energy absorption by Au-urine and Au-cytosine adducts which can then transfer energy to thymine residues. There is no evidence for a "heavy atom" effect which enables metals such as Ag to mix excited states of DNA and to increase the quantum yields of some photoproducts. The use of mid-UV radiation as a probe for investigating the interaction between DNA and drugs such as sodium aurothiomalate is discussed.     

Myelotoxicity of gold.

Of 55 patients who developed blood dyscrasias attributable to gold treatment 15 with bone marrow hypoplasia died. A few of the dyscrasias, occurring in patients who had taken a low total dose of sodium aurothiomalate, may have resulted from immune hypersensitivity, but most, occurring in patients who had taken a higher total dose, were due to cumulative toxicity. All patients receiving gold treatment should undergo frequent blood counts. Any pronounced or continuing fall in the counts is a warning of toxicity, and gold treatment should be stopped. Treatment should be resumed only with caution, and in some patients already in remission lower doses may be just as effective in controlling the disease.     

1H, 13C NMR, and electronic absorption spectroscopic studies of the interaction of cyanide with aurothiomalate

The reaction between cyanide and aurothiomalate (Autm) has been studied by 1H and 13C NMR spectroscopy and by uv spectroscopy. At cyanide:Autm ratios greater than or equal to 2, aurocyanide, [Au(CN)2]-, is the sole product but was also produced at lower ratios. Two intermediates were also identified. These were a mixed ligand complex, [tmAuCN]-, which accounted for over 80% of the gold at a ratio of cyanide to Autm of 1, and a bisthiomalato complex, [Autm2]-, which accounted for 6.8% of the total gold at this ratio of cyanide to Autm. The formation of these complexes may be significant in the antiarthritic activity of Autm since cyanide is produced by potential target cells such as polymorphonuclear leukocytes.     

Energy dispersive X-ray analysis of tissue gold after silver amplification by physical development

Summary Rats were treated intraperitoneally with the gold-containing compounds sodium aurothiomalate (Myocrisin), sodium aurothiosulfate (Sanocrysin), and aurothioglucose. Using stem energy dispersive X-ray analysis, gold and silver were shown to be located at the same point in lysosomes of proximal tubular cells of the kidney, in hepatocytes and in macrophages of lymph glands, spleen and liver. This result indicates that, after exposure to ultraviolet radiation, chemically bound tissue gold is transformed to metallic gold that subsequently can catalyze the reduction of silver ions to silver when subjected to physical development, i.e. exposed to a photographic developer containing silver ions in addition to the reducing molecules.