AM Assay Research Articles

Synthesis, characterization and in vitro cytotoxicity of Co(II) complexes with N6-substituted adenine derivatives: X-ray structures of 6-(4-chlorobenzylamino)purin-di-ium diperchlorate dihydrate and [Co 6(μ-L 6) 4Cl 8(DMSO) 10] · 4DMSO

Cobalt(II) complexes of the composition [Co(L 1)Cl(H 2O) 2] · H 2O ( 1), [Co(L 2)Cl(H 2O) 2] · 2H 2O ( 2), [Co(L 3)Cl(H 2O) 2] · H 2O ( 3), [Co(L 4)Cl(H 2O) 2] · H 2O ( 4), [Co(L 5)Cl(H 2O) 2] · H 2O ( 5) and [Co(L 6)Cl(H 2O) 2] · H 2O ( 6), where HL 1 = 6-(3-chlorobenzylamino)purine, HL 2 = 6-(4-chlorobenzylamino)purine, HL 3 = 6-(2,3-dimethoxybenzylamino)purine, HL 4 = 6-(3,4-dimethoxybenzylamino)purine, HL 5 = 6-(3-fluorobenzylamino)purine and HL 6 = 6-(4-fluorobenzylamino)purine, have been prepared. The compounds have been characterized by elemental analyses, ES+ mass, UV–Vis, IR and NMR spectroscopies, magnetic measurements, molar conductances and thermogravimetric analysis as mononuclear tetrahedral high-spin cobalt(II) complexes. In vitro cytotoxicities of the complexes were tested by a Calcein AM assay against the following human tumour cell lines: malignant melanoma ( G361), chronic myelogenous erythroleukemia ( K562), osteogenic sarcoma ( HOS) and breast adenocarcinoma ( MCF7). The molecular structures of 6-(4-chlorobenzylamino)purin-di-ium diperchlorate dihydrate, [H 2+L 2](ClO 4) 2 · 2H 2O, and a hexanuclear Co(II) complex, [Co 6(μ-L 6) 4Cl 8(DMSO) 10] · 4DMSO ( 7), have been determined by a single crystal X-ray analysis. To elucidate the manner of the organic ligand coordination to cobalt, the geometry of the complex 2 was optimized using DFT calculations at the B3LYP/6-311+G* level of the theory.

Changes in oxidative balance in rat pericytes exposed to diabetic conditions.

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.

Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.

Synthesis, characterization and antitumour activity of copper(II) 6-(4-chlorobenzylamino)purine complexes. X-ray structure of 6-(4-chloro-benzylamino)purinium perchlorate

Copper(II) complexes with 6-(4-chlorobenzylamino)purine (HL) have been prepared and characterized by elemental analysis, electronic, i.r., ES + mass and 13C-n.m.r. spectra, and by magnetic and conductivity data. Based on these physical methods, mainly the temperature dependence of magnetic susceptibility data, we conclude that the complexes are: [Cu2(μ-Cl)2(μ-HL)2Cl2] (1) and [CuCl3(H+L)2]Cl (2). They have been studied for their possible antitumour activity against human malignant melanoma G-361, human osteogenic sarcoma HOS, human chronic myelogenous leukaemia K-562 and human breast adenocarcinoma MCF7 cells in vitro. The IC50 values were determined by calcein AM assay. The molecular structure of 6-(4-chlorobenzylamino)purinium perchlorate, i.e. a protonated form of the free HL ligand, has been determined by a single crystal X-ray analysis.

Deleterious effects of nifedipine on smooth muscle cells implies alterations of intracellular calcium signaling.

Nifedipine (NIF), a calcium channel blocker (CCB) from the first generation of dihydropyridines, induces detrimental effects on patients with cardiovascular diseases. We designed experiments to study, at cellular and molecular level, the mechanisms involved in the induction of deleterious effects by this drug. To this purpose, cultured human smooth muscle cells (HSMC) were used. The effect of NIF and two other CCB (FEL, AML) and inhibitors of intracellular signaling pathways (RR, TG, CAF and GEN) on intracellular calcium [Ca(2+)]I was determined by spectrofluorimetry using Fura 2 AM assay. The results showed that: (i) 10 microM NIF induced the increase of [Ca(2+)]I above the basal values (202.77 +/- 23.98 nM vs. 48.68 +/- 6.45 nM), an effect that was prevented by RR (50.45 +/- 13.9 nM) and was not induced by the two other CCB; (ii) NIF had a thapsigargin-like effect, because it induced the same release of intracellular calcium as TG (212.1 +/- 25.62 nM); (iii) The response to NIF was reduced by 40% after the inhibition of IP3 receptor (121.21 +/- 26.01 nM) and by 50% after the inhibition of tyrosine kinase (101.91 +/- 7.76 nM). Together, these data demonstrate that NIF produces a deregulation of intracellular calcium homeostasis. The abnormal increase of [Ca(2+)]I is due to the activation of store operated channels from the plasma membrane responsible for capacitative calcium entry, a process modulated by the activity of tyrosine kinase and the Ca(2+)-ATPase pump from the sarcoplasmic reticulum.

Metal complexes as anticancer agents 2. Iron(III) and copper(II) bio-active complexes with N6-benzylaminopurine derivatives

Iron(III) complexes with 2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine ( Bohemin, HL 1), in its protonized form, of the composition (H +HL 1) 2[FeCl 5]·2H 2O ( 1), (H +HL 1) 2[FeCl 5]·3H 2O ( 2) have been prepared by two different routes. A new way for synthesis of copper(II) complex with 6-(2-chlorobenzylamino)purine (HL 2), [Cu 2( μ-Cl) 2( μ-HL 2) 2(HL 2) 2Cl 2]·2H 2O ( 3), together with the preparation of copper(II) complex with 6-(3-chlorobenzylamino)purine (HL 3), [Cu 2( μ-Cl) 2( μ-HL 3) 2Cl 2] ( 4), is also reported. The characterization have been based on elemental analysis, electronic, infrared, ES+ mass and 57Fe Mössbauer spectra, conductivity data and magnetic susceptibility temperature measurements over the 4.5–300 K for 1– 3, and 35–300 K for 4, temperature range, respectively. Molecular structure of an electroneutral form of the HL 2 ligand, (HL 2·2H 2O), and a protonized form of the HL 3 ligand, (H +HL 3Cl), have been determined by a single-crystal X-ray analysis. A mononuclear trigonal-bipyramidal (for 1 and 2), binuclear octahedral (for 3) and binuclear trigonal-bipyramidal (for 4) structures of the complexes were proposed mainly on the basis of spectral and magnetic properties. An S=3/2–5/2 spin-admixed state in 1 and 2 was found to be related to the presence of [FeCl 5] 2− ( S=3/2) and [FeCl 5(H 2O)] 2− ( S=5/2) complex anions in 1 and 2, as found by 57Fe Mössbauer spectroscopy. Cytotoxic activity of the complexes was determined by a calcein AM assay and IC 50 values were also estimated. For testing, human malignant melanoma cell line G-361, human osteogenic sarcoma cell line HOS, human chronic myelogenous leukaemia K-562 and human breast adenocarcinoma cell line MCF7 were used. The inhibition of p34 cdc2 kinase by the complexes 1 and 2, which is known to be one of the important mechanisms responsible for cytotoxicity of cytokinin-derived compounds, was also studied.

Preparation, physicochemical properties and biological activity of copper(II) complexes with 6-(2-chlorobenzylamino)purine (HL 1) or 6-(3-chlorobenzylamino)purine (HL 2). The single-crystal X-ray structure of [Cu(H +L 2) 2Cl 3]Cl·2H 2O

Copper(II) complexes of 6-(2-chlorobenzylamino)purine (HL 1) and 6-(3-chlorobenzylamino)purine (HL 2), respectively, were prepared. Depending on the pH of the medium and the molar ratio of reactants the following mononuclear (trigonal-bipyramidal) and dinuclear (octahedral, trigonal-bipyramidal or tetrahedral) complexes were isolated: [Cu 2(μ-HL 1) 2(μ-Cl 2) 2(HL 1) 2Cl 2] ( 1a, b), [Cu 2(μ-Cl) 2(μ-L 1) 2(H 2O) 2] ( 2a), [Cu 2(μ-Cl) 2(μ-L 2) 2(H 2O) 2] ( 2b), [Cu(H +L 2) 2Cl 3]Cl·H 2O ( 3a, b), [Cu 2(μ-Cl) 2(HL 1) 2Cl 2] ( 4a), and [Cu 2(μ-Cl) 2(HL 2) 2Cl 2] ( 4b). The compounds were characterized by elemental analyses, electronic, infrared and mass (FAB+, ES+) spectral data, magnetic susceptibility temperature dependence measurements and molar conductivity data. An X-ray single-crystal structural analysis of [Cu(H +L 2) 2Cl 3]Cl·2H 2O ( 3b) showed that the Cu 2+ ion is penta-coordinated by three chloride ions and by two H +L 2 ligands. Thus, the Cu 2+ ion adopts a distorted trigonal bipyramidal coordination geometry with the protonated H +L 2 ligands coordinated in trans apical positions, while the three chloride ions are situated in an equatorial plane. The cytotoxic activity of the complexes was determined by a calcein AM assay. Mouse melanoma cell line B16-FO, human malignant melanoma cell line G361, human osteogenic sarcoma cell line HOS and human breast adenocarcinoma cell line MCF7 were used. IC 50 values, the drug concentrations lethal to 50% of the tumor cells, were estimated. One of the important mechanisms responsible for the cytotoxicity of cytokinin-derived compounds, the inhibition of cyclin-dependent kinases by the studied complexes, was also determined.

New dedicated AMS system at the Nuclear Regulatory Authority in Argentina

At present, a new AMS system is being installed, at the Nuclear Regulatory Authority, in Argentina. The main goal of the system will be AMS assays of actinides in the area of nuclear safeguards. The installation of the facility started in January 1998. The present status of the project is described.