Influence of ascorbic acid on the activity of the investigational anticancer drug KP1019

Ascorbic acid has been previously discussed to have antitumor potential through its interaction with transition metal ions such as iron and copper. Furthermore, ascorbic acid may act as a reducing agent for Ru(III) compounds such as indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), an investigational anticancer drug which is supposed to be activated by reduction, prior to binding to cellular target proteins. Therefore, we investigated the influence of ascorbic acid on the activity of this antitumor metal complex in cell culture studies. We show that co-incubation of equicytotoxic, constant amounts of KP1019 with high concentrations of ascorbic acid (50–700 μM) increases cytotoxicity of the ruthenium anticancer drug in the human colon carcinoma cell line SW480, human cervical carcinoma KB-3-1 cells, and the multidrug-resistant subline KBC-1, whereas addition of low concentrations (2.7–50 μM) has a strong chemoprotective effect in the human colon carcinoma cell line SW480, but not in multidrug-resistant KBC-1 cells. Although cellular uptake of KP1019 is not altered, ascorbic acid induce stronger interaction of the ruthenium compound with DNA both in SW480 cells and under cell-free conditions with plasmid DNA. Even if DNA interactions probably play a subordinate role in vivo given the extensive protein binding of the compound, our data exemplify that ascorbic acid enhances the reactivity of KP1019 with biomolecules. Moreover, we demonstrate that the levels of KP1019-generated reactive oxygen species are markedly decreased by co-incubation with ascorbic acid. Conclusively, our results indicate that application of high doses of ascorbic acid might increase the anticancer effects of KP1019.     

Chiral ruthenium(II) anthraquinone complexes as dual inhibitors of topoisomerases I and II

Abstract  

DNA topoisomerases (I and II) have been one of the excellent targets in anticancer drug development. Here two chiral ruthenium(II) anthraquinone complexes, Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺, where bpy is 2,2′-bipyridine and ipad is 2-(anthracene-9,10-dione-2-yl)imidazo[4,5-f][1,10]phenanthroline, were synthesized and characterized. As expected, both of the Ru(II) complexes intercalate into DNA base pairs and possess an obviously greater affinity with DNA. Topoisomerase inhibition and DNA strand passage assay confirmed that the two complexes are efficient dual inhibitors of topoisomerases I and II by interference with the DNA religation. In MTT cytotoxicity studies, two Ru(II) complexes exhibited antitumor activity against HeLa, MCF-7, HepG2 and BEL-7402 tumor cell lines. Flow cytometry analysis shows an increase in the percentage of cells with apoptotic morphological features in the sub-G1 phase for Ru(II) complexes. Nuclear chromatin cleavage has also been observed from AO/EB staining assay and alkaline single-cell gel electrophoresis (comet assay). The results demonstrated that Δ- and Λ-[Ru(bpy)₂(ipad)]²⁺ act as dual inhibitors of topoisomerases I and II, and cause DNA damage that can lead to cell cycle arrest and/or cell death by apoptosis.     

Targeting topoisomerase II with the chiral DNA-intercalating ruthenium(II) polypyridyl complexes

Many antitumor drugs act as topoisomerase inhibitors, and the inhibitions are usually related to DNA binding. Here we designed and synthesized DNA-intercalating Ru(II) polypyridyl complexes Δ--[Ru(bpy)₂(uip)]²⁺ and Λ-[Ru(bpy)₂(uip)]²⁺ (bpy is 2,2′-bipyridyl, uip is 2-(5-uracil)-1H-imidazo[4,5-f][1,10]phenanthroline). The DNA binding, photocleavage, topoisomerase inhibition, and cytotoxicity of the complexes were studied. As we expected, the synthesized Ru(II) complexes can intercalate into DNA base pairs and cleave the pBR322 DNA with high activity upon irradiation. The mechanism studies reveal that singlet oxygen (¹O₂) and superoxide anion radical (O₂^•−) may play an important role in the photocleavage. The inhibition of topoisomerases I and II by the Ru(II) complexes has been studied. The results suggest that both complexes are efficient inhibitors towards topoisomerase II by interference with the DNA religation and direct topoisomerase II binding. Both complexes show antitumor activity towards HELA, hepG2, BEL-7402, and CNE-1 tumor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mixed-ligand complexes of ruthenium(II) containing new photoactive or electroactive ligands: synthesis, spectral characterization and DNA interactions

Mixed-ligand ruthenium(II) complexes of three photoactive ligands, viz., (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-naphthyl)-1-ethene (mppne), (E)-1-(9-anthryl)-2-[2-(4-methyl-2-pyridyl)-4-pyridyl]-1-ethene (mppae) and (E)-1-[2-(4-methyl-2-pyridyl)-4-pyridyl]-2-(1-pyrenyl)-1-ethene (mpppe), in which a 2,2′-bipyridyl unit is linked via an ethylinic linkage to either a naphthalene, an anthracene or a pyrene chromophore and three electroactive ligands, viz., 4-(4-pyridyl)-1,2-benzenediol (catpy), 5,6-dihydroxy-1,10-phenanthroline (catphen) and 1,2-benzenediol (cat), were synthesized in good to moderate yields. Complexes [Ru(bpy)₂(mppne)]²⁺ (bpy is 2, 2′–bipyridyl), [Ru(bpy)₂(mppae)]²⁺, [Ru(bpy)₂(mpppe)]²⁺, [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ (phen is 1,10-phenanthroline) were fully characterized by elemental analysis, IR, ¹H NMR, fast-atom bombardment or electron-impact mass, UV–vis and cyclic voltammetric methods. In the latter three complexes, the ligands catpy, catphen and cat are actually bound to the metal center as the corresponding semiquinone species, viz., 4-(4-pyridyl)-1,2-benzenedioleto(+I) (sq-py), 1,10-phenanthroline-5,6-dioleto(+I) (sq-phen) and 1,2-benzenedioleto(+I) (bsq), thus making the overall charge of the complexes formally equal to + 1 in each case. These three complexes are electron paramagnetic resonance active and exhibit an intense absorption band between 941 and 958 nm owing to metal-to-ligand charge transfer (MLCT, d _Ru→π*_sq) transitions. The other three ruthenium(II) complexes containing three photoactive ligands, mppne, mppae and mpppe, exhibit MLCT (d _Ru→π*_bpy ) bands in the 454–461-nm region and are diamagnetic. These can be characterized by the ¹H NMR method. [Ru(bpy)₂(mppne)]²⁺, [Ru(bpy)₂(mppae)]²⁺ and [Ru(bpy)₂(mpppe)]²⁺ exhibit redox waves corresponding to the Ru^III/Ru^II couple along with the expected ligand (bpy and substituted bpy) based ones in their cyclic and differential pulse voltammograms (CH₃CN, 0.1 M tetrabutylammonium hexafluorophosphate)—corresponding voltammograms of [Ru(bpy)₂(sq-py)]⁺, [Ru(bpy)₂(sq-phen)]⁺ and [Ru(phen)₂(bsq)]⁺ are mainly characterized by waves corresponding to the quinone/semiquinone (q/sq) and semiquinone/1,2-diol (sq/cat) redox processes. The results of absorption and fluorescence titration as well as thermal denaturation studies reveal that [Ru(bpy)₂(mppne)]²⁺ and [Ru(bpy)₂(mppae)]²⁺ are moderate-to-strong binders of calf thymus DNA with binding constants ranging from 10⁵ to 10⁶ M⁻¹. Under the identical conditions of drug and light dose, the DNA (supercoiled pBR 322) photocleavage activities of these two complexes follow the order:[Ru(bpy)₂(mppne)]²⁺>[Ru(bpy)₂(mppae)]²⁺, although the emission quantum yields follow the reverse order. The other ruthenium(II) complexes containing the semiquinone-based ligands are found to be nonluminescent and inefficient photocleavage agents of DNA. However, experiments shows that [Ru(bpy)₂(sq)]⁺-based complexes oxidize the sugar unit and could be used as mild oxidants for the sugar moiety of DNA. Possible explanations for these observations are presented.Electronic Supplementary Material Supplementary material is available for this article at .     

Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA,Inhibition of Telomerase Activity and Cellular Uptake

Two ruthenium(II) complexes, Λ-[Ru(phen)₂(p-HPIP)]²⁺ and Δ-[Ru(phen)₂(p-HPIP)]²⁺, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)₂(p-HPIP)]²⁺ against telomerase, suggesting that Λ-[Ru(phen)₂(p-HPIP)]²⁺ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)₂(p-HPIP)]²⁺ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.     

Catalysis of regioselective reduction of NAD+ by ruthenium(II) arene complexes under biologically relevant conditions

Ruthenium(II) arene anticancer complexes [(η ⁶-arene)Ru(en)Cl]PF₆ (arene is hexamethylbenzene, p-cymene, indan; en is ethylenediamine) can catalyse regioselective reduction of NAD⁺ by formate in water to form 1,4-NADH, at pD 7.2, 37 °C, and in the presence of air. The catalytic activity is markedly dependent on the arene, with the hexamethylbenzene (hmb) complex showing the highest activity. For [(η ⁶-hmb)Ru(en)Cl]PF₆, the rate of reaction is independent of NAD⁺ concentration and shows saturation kinetics with respect to formate concentration. A K _m value of 58 mM and a turnover frequency at saturation of 1.46 h⁻¹ were observed. Removal of chloride and performing the reaction under argon led to higher reaction rates. Lung cancer cells (A549) were found to be remarkably tolerant to formate even at millimolar concentrations. The possibility of using ruthenium arene complexes coadministered with formate as catalytic drugs is discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm², respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization. Received: 13 October 2000 / Accepted: 17 November 2000     

Selectivity at a three-base bulge site in the DNA binding of ΔΔ-[{Ru(phen)2}2(μ-dppm)]4+ [dppm is 4,6-bis(2-pyridyl)pyrimidine; phen is 1,10-phenanthroline]

The binding of the stereoisomers of [{Ru(phen)₂}₂(μ-bpm)]⁴⁺, [{Ru(phen)₂}₂(μ-dppm)]⁴⁺ and [{Ru(phen)₂}₂(μ-bb)]⁴⁺ {phen is 1,10-phenanthroline; bpm is 2,2′-bipyrimidine, dppm is 4,6-bis(2-pyridyl)pyrimidine, bb is 1,2-bis[4-(4′-methyl-2,2′-bipyridyl)]ethane} to an oligonucleotide duplex [d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC)] containing a three-base bulge has been studied using a fluorescence intercalator displacement assay. Of the dinuclear ruthenium complexes, the dppm-linked species showed the strongest binding to the oligonucleotide, with the ΔΔ isomer binding slightly more strongly than the meso isomer and the ΛΛ isomer exhibiting the weakest binding. In order to determine whether the ΔΔ-[{Ru(phen)₂}₂(μ-dppm)]⁴⁺ metal complex specifically bound at the three-base bulge site, a ¹H NMR study of the binding of the metal complex to the oligonucleotide duplex d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC) was carried out. Although a detailed picture of the metal complex–oligonucleotide association could not be determined from the NMR results owing to the broadening of the resonances from the metal complex and nucleotide residues at the bulge site, the NMR results do indicate that the metal complex specifically binds at the three-base bulge site. The combined results of this study suggest that the dppm-bridged dinuclear ruthenium complexes have considerable potential as probes for the unusual secondary structure obtained by the insertion of a three-base bulge within duplex DNA.     

Antitumor activity of bent metallocenes: electronic structure analysis using DFT computations

The antitumor activities of bent metallocenes [Cp–M–Cp]²⁺ (M = Ti, V, Nb, Mo) and complexes of them with guanine, adenine, thymine and cytosine nucleotides have been probed using electronic structure calculations. DFT/BP86 calculations have revealed that the bent metallocene–nucleotide interaction strongly depends on the stability of the hydrolyzed form of the bent metallocene dichloride [Cp₂M]²⁺ species, and in turn the stability of the [Cp₂M]²⁺ species strongly depends on the electronic structure of [Cp₂M]²⁺. Detailed electronic structure and Walsh energy analyses have been carried out for the hydrolyzed forms of four [Cp–M–Cp]²⁺ (M = Ti, V, Nb, Mo) species to find out why the bent structure is unusually stable. Energy changes that occur during the bending process in frontier molecular orbitals as well as the p(π)–d(π) overlap have been invoked to account for the anticipated antitumor activities of these species. The bonding situation and the interactions in bent metallocene–nucleotide adducts were elucidated by fragment analysis. Of the four nucleotides complexed with the four bent metallocenes, adenine and guanine show better binding abilities than the other two nucleotides. Metallocenes of second-row transition metals exhibit better binding with pyrimidine-base nucleotides. In particular, the Lewis acidic bent metallocenes interact strongly with nucleotides. The antitumor activity is directly related to the binding strength of the bent metallocene with nucleotide adducts, and the computed interaction energy values correlate very well with the experimentally observed antitumor activities.     

The ruthenium(II)–arene compound RAPTA-C induces apoptosis in EAC cells through mitochondrial and p53–JNK pathways

An investigation of the molecular mechanism of the anticancer activity demonstrated by the ruthenium(II)–arene compound [Ru(η⁶-p-cymene)Cl₂(pta)] (pta is 1,3,5-triaza-7-phosphaadamantane), termed “RAPTA-C”, in Ehrlich ascites carcinoma (EAC) bearing mice is described. RAPTA-C exhibits effective cell growth inhibition by triggering G₂/M phase arrest and apoptosis in cancer cells. Cell cycle arrest is associated with increased levels of p21 and reduced amounts of cyclin E. RAPTA-C treatment also enhances the levels of p53, and its treatment triggers the mitochondrial apoptotic pathway, as shown by the change in Bax to Bcl-2 ratios, resulting in cytochrome c release and caspase-9 activation. c-Jun NH₂-terminal kinase (JNK) is a critical mediator in RAPTA-C-induced cell growth inhibition. Activation of JNK by RAPTA-C increases significantly during apoptosis. Overall, these results suggest a critical role for JNK and p53 in RAPTA-C-induced G₂/M arrest and apoptosis of EAC-bearing mice. Consequently, RAPTA-C treatment results in a significant inhibition in the progression of cancer in an animal model, which emulates the human disease, and does so with remarkably low general toxicity; hence, RAPTA-C has potential for clinical application.     

Benzothiazole bipyridine complexes of ruthenium(II) with cytotoxic activity

A series of benzothiazole-substituted trisbipyridine ruthenium(II) analogues {[Ru(bpy)₂(4,5′-bbtb)]²⁺, [Ru(bpy)₂(5,5′-bbtb)]²⁺ and [Ru(bpy)₂(5-mbtb)]²⁺ [bpy is 2,2′-bipyridine, bbtb is bis(benzothiazol-2-yl)-2,2′-bipyridine, 5-mbtb is 5-(benzothiazol-2-yl),5′-methyl-2,2′-bipyridine]} have been prepared and compared with the complex [Ru(bpy)₂(4,4′-bbtb)]²⁺ reported previously. From the UV–vis spectral studies, substitution at the 5-position of the bpy causes the ligand-centred transitions to occur at considerably lower energy than for those with the functionality at the 4-position, while at the same time causing the emission to be effectively quenched. However, substitution at the 4-position causes the metal-to-ligand charge transfer to occur at lower energies. Fluorescent intercalator displacement studies indicate that the doubly substituted complexes displace ethidium bromide from a range of oligonucleotides, with the greater preference shown for bulge and hairpin sequences by the Λ enantiomer. Since the complexes only show small variation in the UV–vis spectra on the introduction of calf thymus DNA and a small increase in fluorescence they do not appear to be intercalators, but appear to associate within one of the grooves. All of the reported bisbenzothiazole complexes show reasonable cytotoxicity against a range of human cancer cell lines. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of Trace and Major Elements in Bronchoalveolar Lavage Fluid of Mycoplasma Bronchopneumonia in Calves

The aim of this study was to evaluate the reliability and effectiveness of direct determination of trace and major element concentrations in bronchoalveolar lavage fluid samples from Holstein calves with Mycoplasma bronchopneumonia (n = 21) and healthy controls (n = 20). The samples were obtained during bronchoscopy using a standard examination method. A total of 18 elements (aluminum, bromine, calcium, chlorine, chromium, copper, iron, potassium, magnesium, manganese, molybdenum, nickel, phosphorous, sulfur, silicon, strontium, titanium, and zinc) were detected by particle-induced X-ray emission. The average bromine, iron, potassium, magnesium, and phosphorous concentrations were higher in calves with bronchopneumonia than in controls (p < 0.05). They were found to have higher amounts of calcium and zinc, and a higher zinc–copper ratio than that in healthy calves (p < 0.001). Based on the receiver operating characteristics curves, we propose a diagnostic cutoff point for zinc–copper ratio for identification of Mycoplasma pneumonia of 8.676. Our results indicate that assessment of the elemental composition of broncholaveolar lavage fluid is a promising diagnostic tool for Mycoplasma bronchopneumonia.     

Synthesis and anticancer activity of certain mononuclear Ru (II) Complexes

Bis(1,10-phenanthroline/2,2′-bipyridine) ruthenium(II)complexes containing TCP, TTZ OPBI, and BTSC ligands (where, TCP = 1-thiocarbamoyl-3,5-diphenyl-2-pyrazoline,TTZ = 2-(3,5-diphenyl-4,5-dihydropyrazol-1-yl)-4-phenylthiazole, OPBI = 2-hydroxyphenyl benzimidazole and BTSC = benzoin thiosemicarbazone) have been prepared and characterized. The spectral data suggested that the ligands were coordinated with the metal through nitrogen, sulfur and oxygen atoms. The target complexes were tested in vivo for anticancer activity against transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC). All these complexes increased the life span of the EAC-bearing mice, decreased their tumor volume and viable ascitic cell count as well as improved Hb, RBC and WBC counts. These results suggest that the Ru(II) complexes exhibit significant antitumor activity in EAC-bearing mice. It was also observed that the ruthenium complexes protected red blood cells from 2,2′-azo-bis(2-methylpropionamidine) dihydrochloride (AAPH)- induced hemolysis. The inhibitory effect was dose-dependent at a concentration of 20–120 μg/ml.     

Mimicking the electron donor side of Photosystem II in artificial photosynthesis

This review focuses on our recent efforts in synthetic ruthenium–tyrosine–manganese chemistry mimicking the donor side reactions of Photosystem II. Tyrosine and tryptophan residues were linked to ruthenium photosensitizers, which resulted in model complexes for proton-coupled electron transfer from amino acids. A new mechanistic model was proposed and used to design complexes in which the mechanism could be switched between concerted and step-wise proton-coupled electron transfer. Moreover, a manganese dimer linked to a ruthenium complex could be oxidized in three successive steps, from Mn₂^II,II to Mn₂^III,IV by the photo-oxidized ruthenium sensitizer. This was possible thanks to a charge compensating ligand exchange in the manganese complex. Detailed studies of the ligand exchange suggested that at high water concentrations, each oxidation step is coupled to a proton-release of water-derived ligands, analogous to the oxidation steps of the manganese cluster of Photosystem II.     

Ruthenium polypyridyl complexes and their modes of interaction with DNA: Is there a correlation between these interactions and the antitumor activity of the compounds?

Various interaction modes between a group of six ruthenium polypyridyl complexes and DNA have been studied using a number of spectroscopic techniques. Five mononuclear species were selected with formula [Ru(tpy)L₁L₂]⁽²⁻ⁿ⁾⁺, and one closely related dinuclear cation of formula [{Ru(apy)(tpy)}₂{μ-H₂N(CH₂)₆NH₂}]⁴⁺. The ligand tpy is 2,2′:6′,2″-terpyridine and the ligand L₁ is a bidentate ligand, namely, apy (2,2′-azobispyridine), 2-phenylazopyridine, or 2-phenylpyridinylmethylene amine. The ligand L₂ is a labile monodentate ligand, being Cl⁻, H₂O, or CH₃CN. All six species containing a labile L₂ were found to be able to coordinate to the DNA model base 9-ethylguanine by ¹H NMR and mass spectrometry. The dinuclear cationic species, which has no positions available for coordination to a DNA base, was studied for comparison purposes. The interactions between a selection of four representative complexes and calf-thymus DNA were studied by circular and linear dichroism. To explore a possible relation between DNA-binding ability and toxicity, all compounds were screened for anticancer activity in a variety of cancer cell lines, showing in some cases an activity which is comparable to that of cisplatin. Comparison of the details of the compound structures, their DNA binding, and their toxicity allows the exploration of structure–activity relationships that might be used to guide optimization of the activity of agents of this class of compounds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis,antineoplastic and cytotoxic activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(S)₂(K)]²⁺, where S = 1,10-phenanthroline/2,2′-bipyridine and K = 4-OH-btsz, 4-CH₃-btsz, 3,4-di-OCH₃-btsz, 4-OH-binh, 4-CH₃-binh, 3,4-di-OCH₃-binh, were prepared and characterized by elemental analysis, FTIR, ¹H-NMR, and mass spectroscopy. The complexes displayed metal–ligand charge transfer (MLCT) transitions in the visible region. These ligands formed bidentate octahedral ruthenium complexes. The title complexes were evaluated for their in vivo anticancer activity against a transplantable murine tumor cell line, Ehrlisch’s ascites carcinoma (EAC), and in vitro cytotoxic activity against human cancer cell lines Molt 4/C₈ and CEM and murine tumor cell line L1210. The ruthenium complexes showed promising biological activity especially in decreasing tumor volume and viable ascites cell counts. Treatment with these complexes prolonged the life span of mice bearing EAC tumors by 10–52%. In vitro evaluation of these ruthenium complexes revealed cytotoxic activity from 0.21 to 24 μM against Molt 4/C₈, 0.16 to 19 μM aginst CEM, and 0.75 to 32 μM against L1210.     

Relative and combined effects of ethanol and protein deficiency on some hair trace elements

This study was performed in order to analyze the relative and combined effects of ethanol and protein deficiency on hair copper, zinc, manganese, and iron content in four groups of seven animals each which were pair-fed during 8 wk with (1) a nutritionally adequate diet, (2) a 36% (as energy) ethanol-containing isocaloric diet, (3) a 2% protein, isocaloric diet, and (4) a 36% ethanol, 2% protein isocaloric diet, respectively, following the Lieber-DeCarli model, and to analyze the relationship between hair copper, zinc, manganese, and iron content, and the liver and muscle content of these elements. Although there was a trend to higher levels of all the elements analyzed in the the hair of the low-protein fed animals, differences were statistically significant regarding copper and manganese, effects being solely attributable to the low protein diet, not to ethanol. Moreover, hair copper was significantly, inversely related with final weight and weight loss. There were significant relationship between liver zinc and muscle zinc (r=0.57, p=0.002), but not between liver or muscle zinc and hair zinc; no correlations were observed between muscle copper and hair copper, nor between liver manganese and hair manganese. An inverse, statistically significant correlation was observed between liver copper and hair copper (r=−0.39, p<0.05).     

Cytotoxic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-Tetraammine(oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of <Emphasis Type="Italic">Allium cepa</Emphasis>

Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cis-tetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use.     

Trace Elements Analysis of Urine and Hair in Tuberculous Pleurisy

In this study, copper, zinc, magnesium, manganese, selenium, and iron of urine and hair were measured in the patients with tuberculous (TB) pleurisy (n = 24) and in the control group (n = 20). Selenium, magnesium, and zinc of hair were found to be significantly lower in TB pleurisy cases than those in the control group (p < 0.05, p < 0.001, and p < 0.01, respectively). On the contrary, selenium and magnesium of urine were found to be significantly elevated in TB pleurisy cases than those in the control group (p < 0.05 and p < 0.001, respectively). There was no significant difference in the value of manganese and iron between TB pleurisy and the control group (p > 0.05). Copper level were significantly increased in hair and decreased in urine of the patients (p < 0.01). The occurrence of these abnormalities constitutes new information regarding trace elements in TB pleurisy patients. These results may provide an additional disease correlate for assessing TB pleurisy risk.     

NMRD studies on phthalate dioxygenase: evidence for displacement of water on binding substrate

 Water proton T ₁ ^–1 measurements at magnetic fields between 0.01 and 50 MHz [nuclear magnetic relaxation dispersion (NMRD) measurements] have been performed on solutions of phthalate dioxygenase (PDO) reconstituted at the catalytic iron site with copper(II) or manganese(II). The data show evidence of a weakly coordinated water molecule in CuPDO; in the presence of the substrate, phthalate, this water appears to become even less tightly bound, and an additional tightly coordinated water can be detected. In PDO reconstituted with manganese, one tightly coordinated water is detected in the presence and in the absence of phthalate. An attempt is made to reconcile these data with low-temperature near-IR magnetic circular dichroism and X-ray absorption data, which show that PDO reconstituted with iron or cobalt is six-coordinate in the absence of substrate and five-coordinate in the presence of substrate. Received: 24 April 1996 / Accepted: 17 July 1996