Mechanistic Investigation into DNA Modification by a RuII ,RhIII Bimetallic Complex.

Despite significant progress in the treatment of cancer, there remains an urgent need for more effective therapies that also have less impact on patient wellbeing. Photodynamic therapy employs targeted light activation of a photosensitizer in selected tissues, thereby reducing off-target toxicity. Our group previously reported a RuII ,RhIII bimetallic architecture that displays multifunctional covalent photomodification of DNA in the therapeutic window in an oxygen-independent manner, features that are essential for treating deep and hypoxic tumors. Herein, we explore the mechanism by which a new analogue, [(phen)2 Ru(dpp)Rh(phen)Cl2 ]3+ , or RuII -RhIII , interacts with DNA. We established that RuII -RhIII exhibits "light switch" behavior in the presence of DNA, undergoing strong electrostatic interactions that might involve groove binding. Furthermore, these noncovalent interactions play a major role in the covalent photobinding and photocleavage of DNA, which occur according to an oxygen-independent mechanism. Polymerase chain reaction (PCR) revealed that covalent modification of DNA by RuII -RhIII , especially photobinding, is critical to inhibiting amplification, thus suggesting that the complex could exert its toxic activity by interfering with DNA replication in cells. This new structural motif, with phenanthroline at all three terminal ligand positions, has a number of properties that are promising for the continued refinement of photodynamic-therapy strategies.

The metallobiochemistry of ultratrace levels of platinum group elements in the rat.

The use of platinum, palladium and rhodium (Platinum Group Elements - PGEs) and the possibility of exposure to their ultratrace levels is increasing. In fact, the exponential development of metallic PGE-based nanoparticles (<100 nm in size) opens extraordinary perspectives in the areas of electrocatalysts and catalytic converters, magnetic nanopowders, polymer membranes, cancer therapy, coatings, plastics, nanofibres and textiles. Like other metal-based nanoparticles, exposure to PGEs nanoparticles may result in a release of ultratrace amounts of Pt, Pd, Rh ions in the body whose metabolic fate and toxicity still need to be evaluated. Furthermore, PGEs can act as allergic sensitizers by acting as haptens and inducing both type I and IV allergic reactions. In this work we studied the in vivo metabolic patterns of ultratrace levels of potent allergens and sensitizers PGE halogenated salts. (191)Pt, (103)Pd and (101m)Rh radioisotopes were prepared via cyclotron irradiation and used for radiolabelling Na2(191)PtCl4, Na2(103)PdCl4 and Na2(101m)RhCl6 salts. These anionic chlorocomplexes were intraperitoneally injected into rats (114 ng Pt kg(-1) bodyweight; 24 ng Pd kg(-1) b.w.; 16 ng Rh kg(-1) b.w.). At 16 h post-exposure, PGEs were poorly but significantly retained in all tissues analysed. Kidneys, spleen, adrenal gland, liver, pancreas and small intestine were the organs with the highest Pt, Pd, Rh concentrations. In the blood 30-35% of (103)Pd and (191)Pt and 10% of (101m)Rh were recovered in the plasma, mainly bound to albumin and to a less extent to transferrin. The hepatic and renal intracellular distribution showed the highest recovery of (191)Pt, (103)Pd and (101m)Rh in the nuclear fraction (liver) and in the cytosol (kidney). Chromatographic separation and ultrafiltration experiments on kidney and liver cytosols showed the strong ability of biochemical macromolecules to bind (191)Pt, (103)Pd and (101m)Rh, and being responsible for the retention of the three elements in the body. The link to macromolecules is the basis for the sensitizing capacity of PGEs.

Cell-selective biological activity of rhodium metalloinsertors correlates with subcellular localization.

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA.

Catalytic protein modification with dirhodium metallopeptides: specificity in designed and natural systems.

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.

Selective cytotoxicity of rhodium metalloinsertors in mismatch repair-deficient cells.

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents.

Nickel, palladium and rhodium induced IFN-gamma and IL-10 production as assessed by in vitro ELISpot-analysis in contact dermatitis patients.

BACKGROUND: Recent attempts to diminish nickel use in most industrial products have led to an increasing utilization of alternative metal compounds for destinations such as the alloys used in orthopaedics, jewellery and dentistry. The present study was undertaken with the aim to evaluate the potential for an allergic response to nickel, palladium and rhodium on the basis of antigen-specific induction of inflammatory/regulatory cytokines, and to characterize, according to the cytokine profiles, the nature of simultaneous positive patch tests elicited in vivo. Peripheral blood mononuclear cells (PBMC) from 40 patients with different patch test results were kept in short term cultures in the presence of optimized concentrations of NiSO4 x 6H2O, PdCl2 and Rh(CH3COO)2. The production of IFN-gamma and IL-10 elicited by metal compounds were analyzed by the ELISpot assay. RESULTS: We found a specific IFN-gamma response by PBMC upon in vitro stimulation with nickel or palladium in well recognized allergic individuals. All controls with a negative patch test to a metal salt showed an in vitro IL-10 response and not IFN-gamma production when challenged with the same compound. Interestingly, all subjects with positive patch test to both nickel and palladium (group 3) showed an in vitro response characterized by the release of IFN-gamma after nickel stimulation and production of IL-10 in response to palladium. CONCLUSION: These results strongly suggest that the different cytokine profiles elicited in vitro reflect different immune responses which may lead to the control of the allergic responses or to symptomatic allergic contact dermatitis. The development of sensitive and specific in vitro assays based on the determination of the cytokine profiles in response to contact allergens may have important diagnostic and prognostic implications and may prove extremely useful in complementing the diagnostic limits of traditional patch testing.

Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate.

Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells. Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA). ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates. It restored the activity of vinblastine, a P-gp substrate, in causing G2/M arrest in MDR cells. In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells. ABA inhibited the photoaffinity labeling of P-gp by [125I]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp. In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate. Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.

A new metal-binding site for yeast phosphoglycerate kinase as determined by the use of a metal-ATP analog.

Suicide substrate beta, gamma-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the "closed" active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK.3-phospho-D-glycerate-RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes.

The novel use of Rh(I) complexes with naphthyridine ligands and poly(oxyethylene) as antitumorals.

Rh(I) complexes adsorbed on polymers, as a way to improve their transport and solubility properties, were studied as antitumor agents. The binding constants of the complexes to the polymer were evaluated in order to determine the conditions for maximum association to the vehicle. The toxicity of the pure complexes and those bound were determined in vivo using female mice. [Rh(NBD)(2,4N)]CIO4, complex A; where NBD = norbornadiene, (2,4N) = 3,3'-dimethylene-2,2'-di-1,8-naphthyridine, was investigated on primary solid tumors and ascitic tumors. [Rh(NBD)(3,4N)]CIO4, complex B; where (3,4N) = 3,3'-trimethylene-2,2'-di-1,8-naphthyridine, was investigated on ascitic tumors. These Rh(I) complexes appear to be promising drugs because of their solubility in aqueous polymer, which make them easier to handle in comparison with the neutral species. These complexes show a similarity to cisplatin by reducing tumor growth and by increasing the survival life span of mice. Poly (oxyethylene) was used to solubilize these poorly water-soluble compounds and to stabilize the compounds in the solution before injection. These studies suggest that both complexes, A and B, are good candidates for tumor control growth and increase the survival time.

Identification of intrinsic high-level resistance to rare-earth oxides and oxyanions in members of the class Proteobacteria: characterization of tellurite, selenite, and rhodium sesquioxide reduction in Rhodobacter sphaeroides.

We have identified intrinsic high-level resistance (HLR) to tellurite, selenite, and at least 15 other rare-earth oxides and oxyanions in the facultative photoheterotroph Rhodobacter sphaeroides grown either chemoheterotrophically or photoheterotrophically. Other members of the class Proteobacteria, including members of the alpha-2 and alpha-3 phylogenetic subgroups, were also shown to effect the reduction of many of these compounds, although genera from the alpha-1, beta-1, and gamma-3 subgroups did not express HLR to the oxyanions examined. Detailed analyses employing R. sphaeroides have shown that HLR to at least one class of these oxyanions, the tellurite class (e.g., tellurate, tellurite, selenate, selenite, and rhodium sesquioxide), occurred via intracellular oxyanion reduction and resulted in deposition of metal in the cytoplasmic membrane. The concomitant evolution of hydrogen gas from cells grown photoheterotrophically in the presence of these oxyanions was also observed. HLR to tellurite class oxyanions in R. sphaeroides was not affected by exogenous methionine or phosphate but was reduced 40-fold by the addition of cysteine to growth media. In contrast HLR to the periodate class oxyanions (e.g., periodate, siliconate, and siliconite) was inhibited by extracellular PO4(3-) but did not result in metal deposition or gas evolution. Finally, we observed that HLR to arsenate class oxyanions (e.g., arsenate, molybdate, and tungstate) occurred by a third, distinct mechanism, as evidenced by the lack of intracellular metal deposition and hydrogen gas evolution and an insensitivity to extracellular PO4(3-) or cysteine. Examination of a number of R. sphaeroides mutants has determined the obligate requirement for an intact CO2 fixation pathway and the presence of a functional photosynthetic electron transport chain to effect HLR to K2TeO3 under photosynthetic growth conditions, whereas functional cytochromes bc1 and c2 were required under aerobic growth conditions to facilitate HLR. Finally, a purification scheme to recover metals from intact bacterial cells was developed.

Antitumor action of rhodium (I) and iridium (I) complexes.

Several rhodium(I) and iridium(I) complexes displayed different degrees of antitumour activity when tested in mice bearing Ehrlich ascites carcinoma. Rhodium (I) and iridium (I) acetylacetonate derivatives caused a high percentage of cures. The rhodium (I) dimers were particularly interesting, since (bis(cycloocta-1,5-diene)micromicron' dichlorodirhodium(I) [RhCODCl]2) was highly effective, whereas its analogues, bis(bicyclo[2,2,1]hepta-2,5-diene)micromicron'-dichlorodirhodium(I) [RhNBDCl]2) and bis(1,5-hexadiene)micromicron' dichlorodirhodium(I) [RhEDCl]2) were virtually inactive. The absence of significant inhibition of DNA, RNA and protein syntheses in tumour cells found for [RhCODCl]2 at therapeutically active dosages, indicates that this substance has a different mechanism of action from that of cis-dichlorodiammine Pt(II) (cis-PDD). The amount of rhodium found in tumour cells after administration of active [RhCODCl]2 was higher than that for [RhEDCl]2, while the rhodium concentration in the ascitic fluid was much higher for [RhEDCl]2. A mechanism based on the chemical properties of the complexes is tentatively proposed for explaining these findings and selective toxicity for tumour cells.

Hydrophobicity of several rhodium(II) carboxylates correlated with their biologic activity.

Rhodium(II) carboxylates differ greatly in antitumor activity and toxicity depending on the properties of the carboxylate group (methoxyacetate, propionate, butyrate, etc.) involved. The solubility characteristics of rhodium(II) carboxylates correlate well with both the antitumor activity and toxicity that these compounds display. The amount of rhodium which is adsorbed by tumor cells in vitro also correlates with the partition coefficient of the rhodium(II) compounds studied. Survival and toxicity studies show rhodium(II) pentanoate to possess the highest therapeutic index against the Ehrlich ascites tumor strain and also show that lengthening the carboxylate R chain beyond the pentanoate reduces the drugs' therapeutic efficacy.

The metabolism of rhodium(II) acetate in tumor-bearing mice.

Rhodium(II) acetate has been shown to have carcinostatic activity in Swiss mice bearing Ehrlich ascites tumors. For metabolic studies, single therapeutic doses of rhodium(II) [1-14C]acetate that had been given i.p. implantations 3 days previously of 50-fold 10(6) Ehrlich ascites tumor cells. The tissue distribution and excretion of the rhodium (measured by atomic absorption spectrometry) and the acetate (measured by 14C label) were followed at designated time intervals up to 24 hr after injection. Rhodium(II) acetate, a neutral cage complex, breaks down to rhodium and acetate ionic species within 2 hr after i.p. injection, as measured by the rapid exhalation of 14CO2. Both the rhodium and 14C label disappear rapidly from the ascites fluid, with a small but variable amount of each species being incorporated into the tumor cells. Both species were detected mainly in the blood plasma, and the primary organ of deposition was the liver. No measurable quantity of rhodium was found in the brain tissue. During the first 24 hr following drug administration, only 5% rhodium was eliminated in the urine.

Nickel deficiency and nickel-rhodium interaction in chicks.

Nickel deficiency was produced in chicks under near optimal growth conditions. This judgment is based on the finding that chicks fed the experimental diet supplemented with nickel had a very satisfactory growth rate, over 600 g in 4 weeks. To induce nickel deficiency, chicks were raised in plastic cages located inside plastic isolators and were fed diets (containing 2-15 ng of nickel/g) based on dried skim milk, acid-washed ground corn, EDTA-extracted soy protein, and corn oil. In 2 experiments, controls were fed 3 mug of nickel/g as NiCl2-6H2O. In experiment 3, instead of 1 control group 25, 50, 250, and 2,500 ng/g of supplemental dietary nickel as NiCl2-6H2O were each given to separate groups of chicks. Nickel deprivation resulted in: ultrastructural changes in the liver with the most obvious abnormality in the organization of the rough endoplasmic reticulum; altered gross appearance, reduced oxidative ability, and decreased lipid phosphorus in the liver; altered shank skin pigmentation that was associated with a decrease in yellow lipochrome pigments; and lower hematocrits. Deficiency also tended to increase the thickness of the legs and size of the hock; decrease the length:width ratios of the tibias and femurs; and decrease the plasma cholesterol. None of the signs of deficiency were seen in chicks fed diets containing at least 52 ng of nickel/g. In one experiment, a group of birds was fed 50 mug of rhodium/g of diet as (ClRh(NH3)5)SO4 to ascertain whether rhodium is a metabolic antagonist of nickel. Supplemental rhodium increased the hematocrits and liver oxidative ability of both nickel-deficient and -supplemented chicks, and increased total liver lipids, liver lipid phosphorus, and liver cholesterol in the nickel-deficient chicks alone. Rhodium did not increase the signs of nickel deficiency.