Studies on the size and stability of chlorpromazine hydrochloride nanostructures in aqueous solution.

The mean aggregate number (MAN) of the antipsychotic drug chlorpromazine hydrochloride (CPZ) nanostructure was investigated by fluorescence quenching using 9-methylanthracene (9-MA) as the quencher. The method was designed to take advantage of the intrinsic fluorescent properties of CPZ. The validity of this method was supported by the results obtained for the MAN which was determined to be approximately 37 for a solution of 10 mM CPZ in 0.1 M pH 6.5 phosphate buffer. An increase in the aggregate size with increasing drug concentration confirmed the stepwise aggregation theory of CPZ micelle formation. Differential scanning calorimetry was used to examine the effects of concentration on the thermodynamics of micellization. The enthalpy of demicellization increased with increasing CPZ concentration (5-12 mM), suggesting a greater stability of the aggregates at higher concentrations. At amphiphile concentrations higher than 12 mM, a plateau of approximately 10 kJ/mol was observed as the enthalpy of demicellization. Fluorescence lifetime results revealed a two-component system at low CPZ concentration, while data at amphiphile concentrations higher than 12 mM could not be fitted to either single or multi-component lifetime values, suggesting an increase in dispersity in these nanostructures at higher CPZ concentrations. Temperatures higher than 40 degrees C tend to destabilize the larger micelles, and demicellization was observed after approximately 45 degrees C. Changes in osmotic pressure in the presence of dextrose up to 0.3 M had no significant effect on the size of these micellar nanostructures.     

Potentiating effect of chlorpromazine on glyceryl guaiacolate ether induced hemolysis of rat,dog and human blood in vitro

The hemolytic effect of glyceryl guiacolate ether (GGF) with and without chloromazine (CPZ) was studied in vitro on rat, dog and human blood. The lowest concentration of GGE which could produce hemolysis of rat red cells was 0.15 M. The time fpr 50% hemolysis (TH50) of blood depended upon the concentration of drug and dilution of blood. A higher concentration of GGE hemolyzed blood much faster than the lower. There was a progressive increase in the TH50 when 0.15 M GGF was tested on blood samples containing increasing numbers of red cells. CPZ in all cases had its own hemolytic effect at higher concentrations. In this regard rat blood was 10 times more sensitive than dog, and human. A striking potentiating effect of CPZ was observed on the hemolytic effect of GGE. The magnitude of potentiation in all cases was directly related to the concentrations of CPZ. Dog blood was found relatively more sensitive to the hemolytic effect of the combination of CPZ and GGE as compared to the rat and human, which acted alike.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

Differential binding of chlorpromazine to human blood cells: Application of the hygroscopic desorption method

Using a modified hygroscopic desorption method (HDM) the binding of chlorpromazine (CPZ) to human blood cells was investigated in the concentration range from 0.01 to 100 μmol/1. For erythrocytes and ghosts the ratio between cell bound and free drug concentration was constant up to 60 μmol/1 CPZ. Saturable binding, however, was observed for lymphocytes, granulocytes and less pronounced for platelets. In contrast to red cells, CPZ binding to white cells and platelets was strongly dependent on pH. For all blood cells a sharp decrease in binding occurred at drug concentrations higher than 60 μmol/1. This can hardly represent a true saturation of binding sites, since membrane damaging effects occur at these concentrations. Our results suggest that binding of CPZ to erythrocytes represents an interaction at the water-membrane interphase. For the different binding pattern of white cells, the cell organelles, the cytoplasma and the different composition of the membranes might be of importance.     

Mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model

Microglial cells have an essential role in neurodegenerative disorders, such as multiple sclerosis. They are divided into two subgroups: M1 and M2 phenotypes. Mesenchymal stem cells (MSC), with neuroprotective and immunomodulating properties, could improve these diseases. We evaluate the immunomodulating effects of MSC on microglial phenotypes and the improvement of demyelination in a cuprizone (CPZ) model of multiple sclerosis (MS). For inducing the chronic demyelination model, C57BL6 mice were given a diet with 0.2% CPZ (w/w) for 12 weeks. In the MSC group, cells were transplanted into the right lateral ventricle of mice. The expression of targeted genes was assessed by real-time polymerase chain reaction. M1 and M2 microglial phenotypes were assessed by immunohistochemistry of inducible nitric oxide synthase (iNOS) and Arg-1, respectively. Remyelination was studied by luxal fast blue (LFB) staining and electron microscopy (EM). We found that MSC transplantation reduced the expression level of M1-specific messenger RNA (mRNA; iNOS and CD86) but increased the expression level of M2 specific genes (CD206, Arg-1, and CX3CR1) in comparison to the CPZ group. Moreover, cell therapy significantly decreased the M1 marker (iNOS⁺ cells), but M2 marker (Arg-1⁺ cells) significantly increased in comparison with the CPZ group. In addition, MSC treatment significantly increased the CX3CL1 expression level in comparison with the CPZ group and led to improvement in remyelination, which was confirmed by LFB and EM images. The results showed that MSC transplantation increases the M2 and decreases the M1 phenotype in MS. This change was accompanied by decrease in demyelination and axonal injury and indicated that MSCs have a positive effect on MS by modification of microglia cells.     

ESR studies on the effect of cholesterol on chlorpromazine interaction with saturated and unsaturated liposome membranes

In this study, the effects of chlorpromazine (CPZ) on lipid order and motion in saturated (DMPC, DMPG) and unsaturated (SOPC) liposome membranes were investigated by electron spin resonance (ESR) spin labeling technique. We have shown that above the main phase transition temperature of membrane lipids (T(M)), CPZ slightly increases lipid order in membranes without cholesterol, whereas below T(M) it has a strong opposite effect. Addition of 30 mol% of cholesterol into DMPC and SOPC membranes changes significantly the CPZ effects both above and below T(M). Additionally, above T(M), the ordering effect of CPZ on pure SOPC membrane is stronger at pH 7.4 than at pH 9.0, whereas below T(M), as well as in the presence of cholesterol, pH does not seem to play a role in CPZ effect on both membranes. Because of the strong influence of membrane composition on CPZ effect on membranes, the use of cholesterol as a marker of CPZ photosensitized reactions has been discussed.     

Seleno-podophyllotoxin derivatives induce hepatoma SMMC-7721 cell apoptosis through Bax pathway

Podophyllotoxin is a well known anti-tumor chemical, but because of its strong side effects much effort has been paid to reduce cytotoxicity by modifying its structure. Here, we evaluate the anti-tumor activity of a new isolated derivative of podophyllotoxin, 4'-demethyl-4-dehydroxy-4-seleno-phenyl-beta-peltatin-epipodophyllotoxin (CPZ) and find that CPZ can suppress the proliferation of human hepatoma SMMC-7721 cells in a dose- and time-dependent manner. Phase-contrast microscope observation and flow cytometric analysis through PI stains showed that the reagents have strong inhibition of SMMC-7721 cell growth, as the cells were blocked in the G2/M period. Cell apoptosis induced by CPZ was further confirmed by staining with M30 Cytodeath antibody. Rh123 label testing revealed that the mitochondrial membrane potential had been decreased by CPZ treatment. Under the stress of CPZ, cytochrome c was secreted into the cytoplasm by mitochondria, and Bax in cytoplasm was translocated into the mitochondrial membrane. These results suggest that CPZ-induced apoptosis may work through a Bax-dependent pathway.     

pH-dependent effects of chlorpromazine on liposomes and erythrocyte membranes

Chlorpromazine (CPZ) is an amphipathic antipsychotic drug that binds to erythrocytes reaching in this way the central nervous system. CPZ is a basic molecule with pK=8.6. This paper reports on CPZ-induced lysis of red blood cells and liposomes. Haemolysis was tested under hypotonic conditions, in the pH range 5.0-10.0. Cell sensitivity towards CPZ increased with increasing pH. Increasing pH caused also a decrease in the critical micellar concentrations of CPZ. These results are interpreted in terms of a competition between repulsive electrostatic forces and attractive hydrophobic forces, that would act both in pure CPZ and in mixed CPZ-phospholipid micelles. In order to eliminate possible pH effects mediated by red blood cell proteins, experiments were carried out in which CPZ induced release of a fluorescent dye from liposomes (large unilamellar vesicles). The latter observations confirmed that membrane sensitivity towards CPZ was increased at higher pH.     

Changes in erythrocyte morphology induced by imipramine and chlorpromazine

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for lh at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP.     

pH-Dependent Effects of Chlorpromazine on Liposomes and Erythrocyte Membranes

Abstract

Chlorpromazine (CPZ) is an amphipathic antipsychotic drug that binds to erythrocytes reaching in this way the central nervous system. CPZ is a basic molecule with pK = 8.6. This paper reports on CPZ-induced lysis of red blood cells and liposomes. Haemolysis was tested under hypotonic conditions, in the pH range 5.0–10.0. Cell sensitivity towards CPZ increased with increasing pH. Increasing pH caused also a decrease in the critical micellar concentrations of CPZ. These results are interpreted in terms of a competition between repulsive electrostatic forces and attractive hydrophobic forces, that would act both in pure CPZ and in mixed CPZ-phospholipid micelles. In order to eliminate possible pH effects mediated by red blood cell proteins, experiments were carried out in which CPZ induced release of a fluorescent dye from liposomes (large unilamellar vesicles). The latter observations confirmed that membrane sensitivity towards CPZ was increased at higher pH.     

Adenosine 3'':5''-monophosphate content and actions in the division cycle of synchronized HeLa cells

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.     

The Neuroleptic Chlorpromazine Inhibits the Cationic and Stimulates the Anionic Phospholipid Precursor Synthesis in Human Lymphocytes

The widely used neuroleptic drug chlorpromazine (CPZ) influences membrane functions at the levels of ionic channels and receptors as shown. Here we show the effect of short term treatments by CPZ (30 μM), on the nucleotide-containing phospholipid precursors in human lymphocyte primary cultures. During 60 minutes incubation of the cells, the CDP-ethanolamine (CDP-EA) content was only slightly reduced (87 to 76 pmol/10⁶ cells), the amount of CDP-choline (CDP-Ch) was inhibited totally (from 25 to 0 pmol) upon the treatment with 30 μM CPZ under the same conditions. It has been shown earlier, that dCTP can be used as well as CTP for biosynthesis of phospholipids. Thus, the separation of the corresponding ribo- and deoxyribo-liponucleotides was developed. CPZ almost completely inhibited the synthesis of both dCDP-EA and dCDP-Ch under the same conditions The synthesis of the activated liponucleotide precursors, can be measured by incorporation of extracellular 14C-dCyt into both dCDP-EA and dCDP-Ch, as shown earlier. While the cationic deoxyribo-liponucleotide content (dCDP-Ch, dCDP-EA) was decreased, the labelling of the anionic phospholipid precursor dCDP-diacylglycerol (dCDP-DAG) was enhanced several times, it could be labelled only in the presence of CPZ from 14C-dCyd. Thus, a principal disturbance of the membrane phospholipid synthesis is presented (i.e., inhibition of the cationic and enhancement of the anionic dCDP-DAG synthesis). This profound influence on the membrane phospholipids by chlorpromazine, might be the primary effect that contributes to the wide spectrum of CPZ effects on neuronal cells.     

Human cells and cell membrane molecular models are affected in vitro by chlorpromazine

This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 μM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 °C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer.     

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.     

Dose dependent effects on cell cycle checkpoints and DNA repair by bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 μM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway.     

A comparative investigation of the antimitotic action of 2-mercaptoethanol and colcemid on V-79 Chinese hamster cells

Abstract. The current study was performed to characterize the antimitotic action of 2-mercaptoethanol (MET) on mammalian cells.
At concentrations of 2.5 × 10^-2 M, MET arrests V-79 Chinese hamster cells in metaphase. Smaller concentrations (from 5 × 10^-3 M) only produce a mitotic block after several hours, only arresting those mitoses which have gone through one cell cycle in the presence of MET. The accumulation of mitoses by MET is smaller in comparison with colcemid, explained by an effect reducing the number of cells which enter mitosis. In contrast to colcemid, MET-concentrations which do not lead to a mitotic block cause a delay in proliferation. It was shown, by means of the BUdR-labelling method that cells in the presence of colcemid concentrations which arrest mitosis again enter interphase and become polyploid, whereas MET leads to an irreversible arrest of mitosis and does not produce polyploidy in V-79 cells.     

Effects of griseofulvin on mitosis in PtK1 cells

The concentration dependent effects of griseofulvin (GF) on mitosis in PtK₁ cells were studied using a combination of time lapse cinematography and polarization and electron microscopy. Low concentrations of GF (4×10^–5 M) allowed a substantial number of cells to enter and complete an apparently normal mitosis. At higher concentrations of GF (1×10^–4 M and 2.5×10^–4 M) all cells entering mitosis were arrested. Typical c-mitotic chromosome arrays were observed at 1×10^–4 M GF with microtubules present but no spindle formed. At 2.5×10^–4 M GF chromosomes did not orient toward a common center to form a c-mitotic figure, but instead remained in a loosely clustered grouping at the center of the cell. Electron microscopy showed microtubules to be absent but revealed an irregularly shaped electron dense cloud around the centrioles. Quantitative polarization microscopy of metaphase cells perfused with GF showed rapid loss of spindle birefringence after exposure to the drug. Coinciding with loss of birefringence the spindle shrank rapidly with a pronounced shortening of pole to pole distance.     

Calcium and chlorpromazine interactions in rat synaptic plasma membranes. A spin-label and fluorescence probe study.

The effects of calcium and of the psychoactive drug chlorpromazine (CPZ) on the rat synaptic plasma membrane have been studied using two stearic nitroxide spin labels having their doxyl groups in positions 5 and 16 and the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS). The mobility of the 5-doxyl stearic spin label which probes the membrane phospholipids in the vicinity of their polar heads is decreased in the presence of both compounds. Calcium is more efficient in this respect than CPZ. In spite of this qualitative similarity of action, CPZ inhibits the effect of calcium and vice versa. No modification of the 16-doxyl stearic spectrum has been observed even at high calcium or CPZ concentrations. An increase in fluorescence intensity and a blue shift in the emission wavelength of ANS-probed membranes are observed with very low CPZ concentrations (10^?7 to 10^?5m). With higher concentrations, a further intensity increase and a further blue shift are due to direct interaction between ANS and CPZ. Calcium also increases the fluorescence intensity of ANS-labeled membranes in the concentration range 10^?5–10^?2m. As for the spin-label data, the effects of both compounds are mutually competitive. It is concluded that calcium interacts principally with the phospholipid polar heads of this type of membrane. However, the competition with CPZ suggests indirectly that this ion is also bound to membrane proteins. CPZ has an affinity for membrane lipids only at high concentrations. In its pharmacologically active concentration range, it is located preferentially on the membrane proteins.     

Inhibition of tetraphenylphosphonium-induced NMR-visible lipid accumulation in human breast cells by chlorpromazine

The effects of chlorpromazine (CPZ) on the lipid accumulation induced by the cationic lipophilic compound tetraphenylphosphonium chloride (TPP) were examined using proton nuclear magnetic resonance spectroscopy (NMR), lipid extraction and thin layer chromatography (TLC), and electron microscopy (EM). Chlorpromazine at concentrations of 12 or 25 microM significantly reduced the NMR-visible lipid accumulation induced by a 48-h treatment with 6.25 microM TPP in the human breast cell line, HBL-100, without affecting cell viability. TPP caused threefold increases in whole-cell triglyceride levels that were attenuated by the addition of CPZ. Electron micrographs of TPP-treated HBL-100 cells showed that the destruction of mitochondria was accompanied by the accumulation of lipid droplets and myelinoid bodies. The addition of CPZ to TPP-treated cells reduced the occurrence of lipid droplets but not of mitochondrial destruction. Treatment with CPZ, in the presence or absence of TPP, resulted in large cytoplasmic inclusions indicating the inhibition of lysosomal metabolism. The induction and attenuation of NMR-visible lipids in conjunction with concomitant changes in both intracellular lipid droplets and whole-cell triglyceride levels provides evidence that NMR-visible lipids arise from cytoplasmic neutral lipid droplets. The observation that CPZ, a known inhibitor of lysosomal and cytosolic lipid metabolism, attenuates the formation of neutral triglycerides indicates that lysosomal processing may be a major step in the accumulation of NMR-visible lipid in breast cell lines.