Biological relevance of CuFeO2 nanoparticles: Antibacterial and anti-inflammatory activity, genotoxicity, DNA and protein interactions.

Heterometal oxide nanoparticles of bioessential metals are shedding new light to nanoparticle-inspired bioapplications. Pairing bioreactive elements like copper and iron can affect the redox dynamic and biological profile of the nanomaterial. Given the complexity of physicochemical properties, biological activity and toxicity concerns, extensive exploration is demanded, especially when active and less active oxidation states participate as in case of cuprous-ferric delafossite CuFeO2 (copper(I)-iron(III)), a less widespread nanomaterial. In that vein, CuFeO2 nanoparticles were synthesized and biological profile was evaluated in comparison with cuprous oxide (Cu2O NPs) counterpart, an already established antimicrobial agent. Interactions with bacteria, proteins and DNA were examined. Cu2O NPs exhibited stronger antibacterial activity (IC50 < 25 µg/ml) than CuFeO2 NPs (IC50 > 100 µg/ml). In vitro exposure of nanoparticles on plasmid DNA unveiled toxicity in the form of DNA damage for Cu2O and enhanced biocompatibility for CuFeO2 NPs. Genotoxicity estimated by the frequency of sister chromatid exchanges, cytostaticity based on the proliferating rate indices and cytotoxicity based on the mitotic indices at human peripheral lymphocyte cultures were all significantly lower in the case of CuFeO2 NPs. Furthermore, through in vitro albumin denaturation assay, CuFeO2 NPs showed better performance in protein denaturation protection, correlating in superior anti-inflammatory activity than Cu2O and similar to acetylsalicylic acid. Synergy of copper(I)-iron(III) in nanoscale is apparent and gives rise to fruitful bioapplications and perspectives.

Docking analysis targeted to the whole enzyme: an application to the prediction of inhibition of PTP1B by thiomorpholine and thiazolyl derivatives.

PTP1b is a protein tyrosine phosphatase involved in the inactivation of insulin receptor. Since inhibition of PTP1b may prolong the action of the receptor, PTP1b has become a drug target for the treatment of type II diabetes. In the present study, prediction of inhibition using docking analysis targeted specifically to the active or allosteric site was performed on 87 compounds structurally belonging to 10 different groups. Two groups, consisting of 15 thiomorpholine and 10 thiazolyl derivatives exhibiting the best prediction results, were selected for in vitro evaluation. All thiomorpholines showed inhibitory action (with IC50 = 4-45 µΜ, Ki = 2-23 µM), while only three thiazolyl derivatives showed low inhibition (best IC50 = 18 µΜ, Ki = 9 µΜ). However, free binding energy (E) was in accordance with the IC50 values only for some compounds. Docking analysis targeted to the whole enzyme revealed that the compounds exhibiting IC50 values higher than expected could bind to other peripheral sites with lower free energy, Eo, than when bound to the active/allosteric site. A prediction factor, E- (ΣEo × 0.16), which takes into account lower energy binding to peripheral sites, was proposed and was found to correlate well with the IC50 values following an asymmetrical sigmoidal equation with r2 = 0.9692.

Thrombophilic gene polymorphisms and recurrent pregnancy loss in Greek women.

INTRODUCTION: Recurrent pregnancy loss (RPL) is a multifactorial disorder. The aim of this study was the detection of various genetic polymorphisms and their correlation to RPL, in Greek women. METHODS: The impact of 12 thrombophilic polymorphisms was evaluated, among 48 Greek women with a history of RPL, vs 27 healthy parous women. Multiplex PCR and in situ hybridization on nitrocellulose films were performed, to investigate 12 genetic polymorphisms previously reported as risk factors for RPL. RESULTS: Heterozygous FV Leiden, homozygous PAI-1 4G/4G, heterozygous MTHFR C677T, homozygous MTHFR A1298C, as much as the combined thrombophilic genotypes MTHFR 677T + ACE Ι/D, MTHFR 677T/1298C + ACE D/D, ACE I/D + b-fibrinogen -455 G/A, FV HR2 + b-fibrinogen -455 G/A showed a correlation as risk factors for RPL, whereas the rest of the investigated polymorphisms and their combinations did not render statistically significant differences between the two groups in study. CONCLUSION: The results of this study, as well as those of similar studies, concerning the detection of genetic, environmental, and physiological factors underlying RPL, will prove of critical significance in the investigation and treatment of thrombophilic predisposition, in cases of RPL.

The cytogenetic action of ifosfamide, mesna, and their combination on peripheral rabbit lymphocytes: an in vivo/in vitro cytogenetic study.

Ifosfamide (IFO) is an alkylating nitrogen mustard, administrated as an antineoplasmic agent. It is characterized by its intense urotoxic action, leading to hemorrhagic cystitis. This side effect of IFO raises the requirement for the co-administration with sodium 2-sulfanylethanesulfonate (Mesna) aiming to avoid or minimize this effect. IFO and Mesna were administrated separately on rabbit's lymphocytes in vivo, which were later developed in vitro. Cytogenetic markers for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and Mitotic Index were recorded. Mesna's action, in conjunction with IFO reduces the frequency of SCEs, in comparison with the SCEs recordings obtained when IFO is administered alone. In addition to this, when high concentrations of Mesna were administered alone significant reductions of the PRI were noted, than with IFO acting at the same concentration on the lymphocytes. Mesna significantly reduces IFO's genotoxicity, while when administered in high concentrations it acts in an inhibitory fashion on the cytostatic action of the drug.

Distribution of ABO and Rh blood groups in Greece: an update.

The aim of this study was to investigate and evaluate the frequency of the antigens classifying the ABO and Rh blood groups in the Greek population. In this study the 3.5% were first generation immigrants with both their parents immigrants from countries of the USSR, while 1.2% had only one immigrant parent, while the other one was Greek. We compared the frequency of distribution of blood groups ABO and Rh to previous studies conducted at a time before Greece became destination for refugees and immigrants from East and Northeast countries. Blood samples were collected from first year medical students. The frequency of distribution of the ABO and Rh blood groups was slightly differentiated in comparison to previous relevant studies. Significant increase was recorded with respect to the emergence of blood group B in the population investigated, and a considerable reduction was noted in blood group O. In reference to the remaining blood groups, no statistically significant difference was documented. The genetic pool and the genetic inventory of the population residing in Greece have been modified during the last years potentially due to the first generation immigrants. The results of this study could contribute significantly to the National Health System in aiding the prediction of percussions of certain diseases related to blood groups, as well as the requirement for certain blood groups within the blood donation program.

In vitro and in vivo cytogenetic effects of recombinant human erythropoietin on the frequency of sister chromatid exchanges alone or in combination with mitomycin C.

BACKGROUND: Erythropoietin (EPO) is a glycoprotein which has a main property, erythropoiesis, but its range of action in the human body is very wide. It has been suggested that EPO acts cytoprotectively for many cell lines against many toxic causes in vitro and in vivo. Our aim was to study the action of EPO on DNA of two cell types, human lymphocytes in vitro and on P388 ascites tumor cells inoculated in BDF1 mice in the presence and absence of the genotoxic agent mitomycin C (MMC). METHOD: The sister chromatid exchange (SCE) assay was used as it is a very sensitive, simple and rapid method for detecting DNA damage. Proliferation rate indices (PRI) and mitotic indices (MI) were also counted. RESULTS: EPO did not alter the SCE level when it acted alone on both cell lines. MMC as a potent genotoxic agent increased SCE levels in vitro and in vivo. EPO used in combination with MMC significantly decreased SCE levels and increased PRI and MI values induced by MMC alone both in vitro and in vivo. CONCLUSIONS: EPO acts protectively against the genotoxic potential of MMC, and this action may have clinical implications.

Prognostic and predictive factors of invasive ductal breast carcinomas.

PURPOSE: To investigate the significance of certain immunohistochemical markers, namely estrogen (ER) and progesterone receptors (PgR), c-erbB-2 oncogene, p53 tumor suppressor gene and E-cadherin adhesion molecule, in invasive ductal breast carcinomas. METHODS: A series of 102 primary breast carcinomas of the ductal type and a standard immunohistochemical technique was used to detect the aforementioned biological markers. The findings were related to various clinical and pathological tumor characteristics, including lymph node metastases. RESULTS: ER and E-cadherin were expressed more commonly in tumors of low histological grade and small number (< or =3) of metastatic lymph nodes, whereas c-erbB-2 and the p53 gene were usually expressed in breast tumors of high histological grade and increased number (>3) of metastatic lymph nodes. PgR, on the other hand, was detected frequently in patients with early menarche and metastases in <3 lymph nodes, but this tendency was not statistically significant. CONCLUSION: The use of these biomarkers, preferably in combination, may provide additional prognostic and therapeutic information which may be proved useful in planning breast cancer treatment.

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters.

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

Cytogenetic study in cultured human lymphocytes treated with three commonly used preservatives.

Potassium sorbate, sodium benzoate and potassium nitrate have been tested for their genotoxic, cytostatic and cytotoxic potential in human peripheral blood cells in vitro. Potassium nitrate has shown no activity in the test system. When potassium sorbate and sodium benzoate were used at concentrations of 2.0, 0.2 and 0.02 mM no cytostatic activity was detected. However, concentrations of 4 and 8 mM have shown a weak cytostaticity. Additionally, a genotoxic activity using the SCE methodology has been observed at 8 mM of sodium benzoate and at 4 and 8 mM of potassium sorbate. No cytotoxic activity has been induced by the three preservatives. Data demonstrate that the preservatives at low concentrations can be considered as non genotoxic under conditions tested.

Comparison of new nitrosoureas esters with modified steroidal nucleus for cytogenetic and antineoplastic activity.

Nitrosourea is decomposed under physiological conditions to react with biological macromolecules by two mechanisms: alkylation (with proteins and nucleic acids) and carbamoylation (with proteins but not nucleic acids). It has been suggested that the alkylating action is responsible for the therapeutic effects of nitrosoureas, and that the carbamoylation activity leads to toxicity effects. In order to reduce systemic toxicity and improve specificity and distribution for cancer therapy, 2-haloethyl nitrosourea has been esterified with modified steroids, which are used as biological platforms for transporting the alkylating agent to the tumor site in a specific manner. The cytogenetic and antineoplastic effect were studied of seven newly synthesized esters of N,N-bis(2-chloroethyl)alanyl carboxyl derivatives with a modified steroidal nucleus (compounds 1-7). As a very sensitive indicator of genotoxicity the Sister Chromatid Exchange (SCE) assay was used and as a valuable marker of cytostatic activity the cell Proliferation Rate Index (PRI) in cultures of normal human lymphocytes was used. The order of magnitude of the cytogenetic activity on a molar basis (15, 30, 120 microM) of the compounds was 7>>6>3>5>2>4>1. The most active compound 7 has an enlarged (seven carbon atoms) A ring modified with a lactam group (-NHCO-) with the nitrosourea moiety esterified at position 17 In the group of seven substances a correlation was observed between the magnitude of SCE response and the depression in PRI (r=-O, 65, p<0.001). According to the criterion of activity of National Cancer Institute (NCI), the order of antineoplastic activity of compounds on lymphoid L1210 leukemia is 7>6>2>5>4>3>1 and on lympocytic P388 leukemia cells is 7>2>6>5>4>3>1. The present results are in agreement with previous suggestions that the effectiveness in cytogenetic activity may well be correlated with antitumor effects [T/C: 248% for the compound 7 in 250 mg/kg b.w.; T/C: mean survival time of drug-treated animals (T) (excluding long term survivals) vs. corn-oil-treated controls (C)].

A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids.

In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer.

Induction of cytogenetic damage in human lymphocytes in vitro and of antineoplastic effects in Ehrlich ascites tumor cells in vivo treated by methotrexate, hyperthermia and/or caffeine.

The synergistic effect of methotrexate (at concentrations between 3. 1 and 100 nM) and its combinations with caffeine (618 microM) and/or hyperthermia (42 degreesC for 2 h) on the frequency of sister chromatid exchanges (SCEs), the proliferating rate index and the mitotic index in cultured human lymphocytes, was examined. Also, the in vivo antineoplastic effects of methotrexate (at a concentration of 0.45 microg/g body weight) and its combination with caffeine (120 microg/g body weight), both on the survival time and the increase of the weight of tumor of BALB/c mice inoculated with Ehrlich ascites tumor cells was examined in the present study. The results indicated that: (a) the triple combination of methotrexate, caffeine and hyperthermia synergistically increased the levels of SCEs and exerted cytostatic and cytotoxic action and (b) the combination of methotrexate and caffeine significantly increased the survival span of the mice inoculated with Ehrlich ascites tumor cells and reduced the increase of the weight of their tumors at rates higher than in the case of methotrexate by itself. It is suggested that the above triple combination (methotrexate plus caffeine plus hyperthermia) could achieve increased effectiveness of methotrexate, better therapy results, and could be successfully applied in the treatment of various types of cancer.

Bis(acetato)bis(1-methyl-4,5-diphenylimidazole)copper(II): preparation, characterization, crystal structure, DNA strand breakage and cytogenetic effect.

The preparation, characterization and antitumour properties of the complex [Cu(O2CMe)2L2] (1), where L = 1-methyl-4,5-diphenylimidazole, are described. The crystal structure of 1 (triclinic, space group P1, a = 6.743(1), b = 8.006(1), c = 15.898(1) A, alpha = 102.87(1), beta = 101.10(1), gamma = 76.76(1) degree, Z = 1) has been determined (R = 0.0254, Rw = 0.0275). In the centrosymmetric complex the copper ion is in an essentially square planar environment consisting of two pyridine-type imidazole nitrogen atoms and an oxygen atom from each acetate ligand; the second oxygen atoms of the carboxylate functionalities are involved in weak interactions with the metal completing the coordination to a very distorted tetragonal bipyramid. Complex 1 has been also characterized by elemental analyses, thermal methods, variable-temperature magnetic susceptibility and spectroscopic (IR and far-IR, FT-Raman, UV/VIS, EPR) techniques. The effect of the complex on the in vitro DNA strand breakage was examined. It was found that 1 causes degradation on the linearized pKS DNA, ds and ss DNA. High concentrations of this Cu(II) complex cause scissions on the relaxed and the supercoiled DNA. Furthermore, the in vivo cytogenic effect of 1 was examined on human lymphocyte cells. This study presents indications that 1 could have some relevance in the treatment of tumour cell lines. An orbital interpretation of the interaction of 1 with the DNA bases is proposed.

Chlorpromazine-induced damage on nucleic acids: a combined cytogenetic and biochemical study.

Chlorpromazine is now emerging as an adjuvant chemotherapeutic agent for the treatment of neoplasia. This was further supported in the present study by the following lines of evidence: it was shown that chlorpromazine causes damage in a series of native nucleic acids, though at somewhat high concentrations. Furthermore, chlorpromazine and caffeine were shown to act synergistically to potentiate the cytogenetic effect of adriamycin on human lymphocytes in vitro and on Ehrlich ascites tumour (EAT) cells in vivo. It is suggested that chlorpromazine alone or in combination with caffeine may exert its cytotoxic effect on normal and neoplastic cells not only indirectly, i.e. by facilitating the intracellular retention of adriamycin, but also directly by intercalating into nucleic acids.

Cytogenetic study for possible mutagenic activity induced by ice-nucleation bacteria or their metabolic products in human lymphocytes in vitro.

A means for eliminating ice-nucleation-active (INA) bacteria, the microorganisms responsible for frost damage to plants at mild freezing temperatures, is the use as competitors of other naturally occurring, non-nucleating strains. Inactive mutants (INA-) of INA bacteria have been produced by genetic or chemical methods and proposed for biological control of INA populations. Since, however, the application of these INA- mutants in the field may create health hazards to animals, we have studied the possible mutagenic activity of the INA- mutants by examining chromosome aberrations, sister-chromatid exchange (SCE) frequencies, and proliferation kinetics of human lymphocyte cultures. These cultures were treated with: (a) a naturally occurring INA- bacterium (p 767), (b) 2 parental strains (cit 7 and cit 13) of INA bacteria isolated from Citrus orchards, and (c) 2 INA- mutant strains (cit 7 del 1b and cit 13-12), produced, respectively, by chemical modification and by deletion of the corresponding parental strains. Neither whole bacteria nor infiltrates of bacterial growth media, in which toxic metabolic bacterial products might have been released, induced elevation of either chromosome aberrations and SCEs or a cell-division delay. Negative results were also obtained when sonicated bacteria were tested for possible intracellular mutagenic components.

Comparative study on cytogenetic effects by diplatinum complexes of the ligands of naphthazarine and squaric acid in human lymphocytes.

The effect of diplatinum complexes of the binucleating ligands of naphthazarine and squaric acid on Sister Chromatid Exchange (SCE) rates and human lymphocyte proliferation kinetics was studied. Squarodicisplatinum complex I, naphthazarindicisplatinum and squarodicisplatinum complex II induce cytotoxic effects as can be deduced from the resulted induction of SCEs and the produced cell division delays. Squarodicisplatinum complex I was found to be on a molar basis the most effective in causing markedly increased SCE rates and cell division delays. Cis-diaminodichloride platinum was found to be next in order of effectiveness with naphthazarindicisplatinum and squarodicisplatinum complex II following. Naphthazarine and SQA were found to be ineffective on induction of SCEs.

Synergistic induction of cytogenetic damage by alkylating antineoplastics and 5-azacytidine in human lymphocytes.

We studied the effects of the inhibitor of DNA methylation, 5-azacytidine (Aza-C), alone and in combination with three antitumor alkylating agents, on sister chromatid exchanges (SCEs) and lymphocyte proliferation kinetics. Aza-C was found to act synergistically on induction of SCEs when administered in combination with either melphalan (MEL) or chlorambucil (CBC) or cis-platinum-(II)diamine dichloride (cis-Pt). Cell-division delays were consistently observed in cultures treated with each of the antineoplastics when introduced concomitantly with Aza-C, compared with cultures treated with antineoplastics alone. Mitotic indices (MI) in cultures treated with each of the three alkylating agents were found to be suppressed by Aza-C. These results support the premise that hypomethylation of DNA causes cytotoxic effects by interfering with DNA repair mechanisms and by inhibiting DNA synthesis, or other cell growth mechanisms, which human lymphocytes undertake after being damaged by alkylation.

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine.

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.

Enhancement and attenuation of cytogenetic damage by vitamin C in cultured human lymphocytes exposed to thiotepa or L-ethionine.

Vitamin C (vit C) at 2 mM enhanced sister chromatid exchange (SCE) frequencies induced by Thiotepa (THIO) or L-ethionine (L-ETH) in cultured human lymphocytes. However, when vit C was tested at 0.02 mM and 0.2 mM a rather protective effect on SCE rates induced by THIO or L-ETH was identified. Vit C (2 mM) caused a cell division delay in cultures treated with THIO or L-ETH. Division delays caused by THIO or L-ETH were reversed in the presence of 0.02 mM or 0.2 mM vit C. Mitotic indices (MIs) in cultures treated with THIO or L-ETH continued to be suppressed in the presence of 2 mM vit C. However, vit C at 0.02 mM reversed suppression of MIs caused by L-ETH or THIO. These findings illustrate the complexity of the interactions of vit C in biological systems and indicate that with different concentrations vit C can cause or prevent genetic toxicity.