Planarians as models to investigate the bioactivity of gold(I) complexes in vivo.

Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.

Sensitization potential of gold sodium thiosulfate in mice and guinea pigs.

Since gold sodium thiosulfate (GST) has been included in a standard patch test series for diagnosis of allergic contact dermatitis from gold, the incidence of patients showing positive reactions to gold is increasing. However, there were little reports on induction of gold sensitization in animals. In this study, we have examined the sensitization potential of GST using mice and guinea pigs. In the guinea pig maximization test, 2 or 6 out of 10 animals showed positive skin responses, mainly edema, by challenge with 2% or 5% GST in 50% ethanol solution, respectively. In the mouse ear swelling test, positive ear swelling (20% greater increase in ear thickness) after challenge with GST was shown in 2 out of 6 mice those previously treated with GST. Topical exposure of mice to GST in 70% dimethylsulfoxide solution induced small increases in the lymph node weight and the lymph node cell (LNC) number in the murine local lymph node assay (LLNA). A greater degree of LNC responses were observed in the sensitive mouse lymph node assay (SLNA) compared with the LLNA, but the stimulation index of total lymph node response by GST was not so high. From these results, GST was identified as a contact allergen, but the sensitization potential was not so strong. In the mouse IgE test, treatment of mice with GST resulted in a statistically significant increase in the serum IgE antibody concentration that associated with immediate-type hypersensitivity reaction. It may suggest that the sensitization responses from gold would appear not only at the contact site but also systematically.

Prevalence of gold sensitivity in asymptomatic individuals with gold dental restorations.

BACKGROUND: The clinical relevance of positive patch test reactions to gold sodium thiosulfate in asymptomatic individuals with gold dental restorations is often unclear. Knowledge of the prevalence of gold sensitivity in individuals with and without gold dental restorations is required to better understand the relevance of these reactions. OBJECTIVE: To determine the prevalence of positive patch test reactions to gold in asymptomatic individuals with gold dental restorations (gold patients) compared with similar individuals without gold dental appliances (nongold patients). METHODS: One hundred thirty-six healthy, asymptomatic patients were patch tested to gold sodium thiosulfate, nickel sulfate and palladium chloride. Readings occurred after 2 days and 7 days. RESULTS: Of the patients tested, 24 of 71 (33.8%) gold patients had a positive reaction to gold versus 7 of 65 (10.8%) of the nongold patients, P <.001. Of those with a positive gold reaction, 12 of 31 (38.7%) also had a positive nickel reaction. Nickel alone was positive in 18 of 71 (25. 4%) of gold patients versus 11 of 65 (16.9%) of nongold patients. 19 of 29 (65.5%) of those with a positive nickel reaction also reacted to palladium and 19 of 22 (86.4%) of those with a palladium reaction also reacted to nickel. The rate of allergy to gold computed over a 3-year period for patients patch tested in the Oregon Health Sciences University (OHSU) Contact Dermatitis Clinic was 13.5% (46/342). CONCLUSIONS: The prevalence of gold sensitivity in individuals with gold dental restorations is approximately 33.8%. This is significantly greater than the 10.8% prevalence seen in individuals without gold dental appliances, as well as greater than the 3-year rate from the OHSU Contact Dermatitis Clinic. This data should help shed light on issues of clinical relevance.

Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C.

Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (PKC) is involved in T cell activation, the capacity of gold compounds to inhibit PKC partially purified from Jurkat cells was assayed in vitro. GST was found to inhibit PKC in a dose-dependent manner, but AUR caused no significant inhibition of PKC at pharmacologically relevant concentrations. The inhibitory effect of GST on PKC was abolished by 2-mercaptoethanol. To investigate the effect of GST on the regulation of PKC in vivo, the levels of PKC activity in Jurkat cells were examined. Cytosolic PKC activity decreased slowly in a concentration- and time-dependent manner as a result of incubation of Jurkat cells with GST. To ascertain whether GST inhibited PKC translocation and down-regulation, PKC activities associated with the membrane and cystosolic fractions were evaluated after phorbol myristate acetate (PMA) stimulation of GST incubated Jurkat cells. Translocation of PKC was markedly inhibited by pretreatment of Jurkat cells with GST for 3 d, but the capacity of PMA to down-regulate PKC activity in Jurkat cells was not altered by GST preincubation. The functional impact of GST-mediated downregulation of PKC in Jurkat cells was examined by analyzing PMA-stimulated phosphorylation of CD3. Although GST preincubated Jurkat cells exhibited an increased density of CD3, PMA-stimulated phosphorylation of the gamma chain of CD3 was markedly inhibited. Specificity for the inhibitory effect of GST on PKC was suggested by the finding that GST did not alter the mitogen-induced increases in inositol trisphosphate levels in Jurkat cells. Finally, the mechanism of the GST-induced inhibition of PKC was examined in detail, using purified PKC subspecies from rat brain. GST inhibited type II PKC more effectively than type III PKC, and also inhibited the enzymatic activity of the isolated catalytic fragment of PKC. The inhibitory effect of GST on PKC activity could not be explained by competition with phospholipid or nonspecific interference with the substrate. These data suggest that the immunomodulatory effects of GST may result from its capacity to inhibit PKC activity.

Contribution to the mode of action of univalent gold.

Au+ binds to the collagen structure and, therefore, the authors studied the kinetics of the interaction between sodium gold thiosulphate (SGTS) and C1q complement subcomponent, the structure of is partially collagenous. The kinetics was evaluated densitometrically and compared with that of collagen and SGTS. On the basis of those results, the effect of SGTS upon collagen type-II-induced arthritis (CIA) was investigated in Wistar rats. SGTS (20 mg per kg of body weight weekly) was administered i.m. starting i) with the first immunization dose, ii) with the second immunization dose, and iii) after the onset of arthritis. Regardless of the timing of drug administration the manifestation of arthritis was decreased, but the decrease was more expressed in the first two groups. It was concluded that SGTS administration is capable of inhibiting CIA, provided that the drug is applied sufficiently early. Since formation of antibodies to type-II collagen remained unaffected, it is feasible that the mode of action of gold complexes is based mainly on blocking the activation of complement system.

Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

Mechanism of inhibition of human neutrophil collagenase by Gold(I) chrysotherapeutic compounds. Interaction at a heavy metal binding site.

The mechanism of inhibition of two forms of human neutrophil collagenase (HNC) by six Au(I) compounds, some of which are used as chrysotherapeutic agents, has been investigated. The two forms of enzyme studied are active and latent HNC, the latter of which is activated by p-chloromercuribenzoate (PCMB). The effects of PCMB and Zn(II), which are normally included in the assays, on the activity of both forms of HNC and on their inhibition by these Au(I) compounds have also been studied. Zn(II) stimulates the activity of both the active and PCMB-activated latent forms of HNC up to a concentration of 50-100 microM, after which it inhibits markedly. PCMB activates latent HNC up to a concentration of 100 microM followed by inhibition at higher concentrations. Active HNC is not stimulated at PCMB concentrations below 100 microM, but is inhibited at higher concentrations. The stimulatory effects of Zn(II) and PCMB on HNC and its inhibition by PCMB are all attributable to binding at distinct sites. The inhibition of both active and PCMB-activated latent HNC by the Au(I) compounds is noncompetitive and is reversed by Zn(II). The inhibition of both forms of HNC by SKF 80544 and SKF 36914, which do not contain thiol ligands, is weak to moderate and is not influenced by the PCMB concentration. In contrast, PCMB markedly enhances the inhibition by Myocrisin, Sanocrisin, and Solganol by complexing to their thiol ligands to facilitate release of the Au(I) atom for binding to HNC. Cd(II) and Cu(II) also inhibit HNC noncompetitively, and inhibition is also reversed by Zn(II). Collectively, these data indicate that latent HNC contains a heavy metal binding site distinct from the active site at which Au(I), Cd(II), and Cu(II) bind to cause noncompetitive inhibition. Occupancy of this site by Zn(II) is characterized by retention of activity.

Effect of two gold compounds on lysosomes.

The effect of two gold(I) compounds on stability of lysosomes in vitro was studied. Lysosomes from homogenates of rat kidney cortex were isolated by differential centrifugation. These lysosomes were incubated at 37 degrees C with widely varied concentrations of sodium aurothiomalate and sodium aurothiosulphate for 5, 35, and 65 minutes. Acid phosphatase activities were measured and used as an indication of lysosomal membrane stability in the presence and absence of drugs. The enhanced release of acid phosphatase from lysosomes by aurothiomalate and aurothiosulphate was related to dose, but the drugs differed substantially in their potencies. The disruptive effect on lysosomes was more marked for aurothiosulphate than for aurothiomalate. In addition, both drugs inhibited acid phosphatase activities at relatively high gold concentrations. Aurothiomalate had a moderate and aurothiosulphate a weaker inhibitory effect on the enzyme. Our results indicate that aurothiomalate and aurothiosulphate exert their beneficial effect in the treatment of rheumatoid arthritis through mechanism(s) other than lysosomal membrane stabilisation.

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy.

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.

Cellular immunity to the mouse pneumonitis agent.

During infection with the mouse pneumonitis biovar of Chlamydia trachomatis, heterozygous (nu/+) mice with relatively intact T cell function develop both delayed hypersensitivity to C trachomatis antigen and antigen-specific lymphocyte transformation, whereas athymic nude (nu/nu) mice do not. Nu/nu mice are protected against death from mouse pneumonitis by transfer of immune T cells from nu/+ mice, which are more resistant to C trachomatis. While this enables athymic mice to make antibody to C trachomatis (which does not occur without reconstitution), resistance correlates best with development of antigen-specific lymphocyte transformation in the recipient animals. During infection nu/+ mice develop activated alveolar macrophages (by both morphological and functional criteria) while nu/nu mice do not. Nu/+ mice that have been preinfected with Histoplasma capsulatum to activate cellular immunity become more resistant to C trachomatis than do nu/+ controls. Cell-mediated immunity to C trachomatis pneumonia is T cell dependent and is important in host defense.