Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide.

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H(2)O(2)) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H(2)O(2) alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 micromol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 micromol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.

Analysis of aclarubicin-induced cell death in human fibroblasts.

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.

The combined effect of IDA and glutaraldehyde on the erythrocyte membrane proteins.

A number of investigators have been focusing their attention on the encapsulation of antineoplastic drugs within erythrocytes to diminish their side-effects. Glutaraldehyde is often used as crosslinking agent to link the drugs (including idarubicin, IDA) to the cells. The previous studies indicated that in glutaraldehyde-treated human erythrocytes the elevated level of drugs was observed but also the various changes in the organization of the red cells were noted. In this study, we continue our investigations on the interaction of IDA and glutaraldehyde on the erythrocytes and now we concentrate on the effect of these compounds with the erythrocyte membrane proteins. For this purpose, SDS-gel electrophoresis of the cell proteins was carried out. Additionally, analysis of the disturbances of erythrocytes shape and size, accompanied by the application of flow cytometry and microscopy examination, were undertaken. The fluorimetric method was used to estimate content of IDA in supernatants, after erythrocyte membranes incubation with different glutaraldehyde concentrations. It was observed that glutaraldehyde caused in gradually dependent manner an increase of percent of IDA linked to the cell membrane proteins. After this incorporation, perturbations in the content of the proteins in the cell membrane were observed. The protein aggregates and changes in the level of spectrin, band 3 protein and small mass proteins were noted. The use of flow cytometry and microscopy technique demonstrated also disturbances in the shape and size of erythrocytes. For all tested concentrations of glutaraldehyde, the changes were statistically significant.

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: relation to drug cytotoxicity and caspase-3 activation.

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.

The influence of 2-chlorodeoxyadenosine (2-CdA) on the adenine energy charge and glutathione content of human erythrocytes.

We have examined the effect of exposure of human erythrocytes to the new chemotherapy drug 2-chlorodeoxyadenosine (2-CdA, cladribine), focusing on the glutathione (GSH and GSSG) content and the adenine energy charge (AEC). Incubation of erythrocytes with 0.1-5 microg/ml 2-CdA induced no significant change in the reduced or total glutathione level or in the AMP and ATP concentrations. The ADP concentration increased slightly and the AEC value is in the range typical of healthy organisms. Incubation of erythrocytes with 2-CdA also caused cell shape changes, converting most of the cells to echinocytes.

Influence of 2-chlorodeoxyadenosine (cladribine) on human erythrocytes.

2-Chlorodeoxyadenosine (2-CdA, cladribine) is one of the newest chemotherapy drugs which has been around and in use for a few years. Drug in tumour cells causes the inhibition of DNA synthesis and repair processes in replication cells, and the accumulation of DNA strand breaks in nonproliferating cells. The present study was undertaken to characterize the influence of cladribine on the fluidity of the lipid bilayer and protein conformation in human erythrocytes. The effect of cladribine on the erythrocyte membrane structure was examined by electron spin resonance (ESR) spectroscopy and fluorescence measurements. It was observed that under the studied conditions (c: 0.1-5 microg/ml, t = 1 h, 37 degrees C), cladribine localised mainly in the erythrocyte membrane and affected its organization. The alterations in the fluidity were observed mainly in the deeper regions of the cell membrane. The incorporation of drug into human erythrocytes also caused negligible conformational alterations of membrane cytoskeletal proteins and did not change the internal viscosity of the cells. We can conclude from these data that 2-CdA in vitro is significantly much less toxic to erythrocytes than anthracycline drugs, which are used in treatment of leukemias. However, the higher concentrations of 2-CdA (about 5 microg/ml) can be also toxic to erythrocytes.

The combined effect of IDA and glutaraldehyde on the properties of human erythrocytes.

The effect of IDA and glutaraldehyde on the properties of human erythrocytes was examined by Electron Spin Resonance spectroscopy and fluorescence measurements. In this study glutaraldehyde was used as the agent linking the drug to the erythrocyte membrane. We have demonstrated that idarubicin (IDA) alone caused only negligible changes of the membrane fluidity. When IDA preincubated erythrocytes were treated with glutaraldehyde, the alterations in the fluidity were observed in the polar parts as well as in the deeper regions of the cell membrane. The incorporation of drug and glutaraldehyde into human erythrocytes also caused conformational alterations of membrane cytoskeletal proteins and changes in the internal viscosity of the cells. Our data suggest that glutaraldehyde in idarubicin-pretreated erythrocytes may potentiate the drug toxicity leading to significant perturbations in the organization of the plasma membrane lipids and proteins.

Cytotoxicity of daunorubicin in trisomic (+21) human fibroblasts: relation to drug uptake and cell membrane fluidity.

The influence of daunorubicin (DNR) on survival of human normal (S-126) and trisomic, with respect to chromosome 21 (T-164; S-240), skin fibroblasts and some parameters related to it, such as intracellular drug accumulation, distribution and interaction with cell membrane, were studied. The in vitro growth-inhibition assay indicated that DNR was less cytotoxic for trisomic than for normal cells. Comparison of kinetic parameters and intracellular distribution of this compound showed that the uptake and the amount of intracellular free DNR were greater in normal than in trisomic cells. Contrary to this, there were no significant differences between the amount of DNA-bound drug in both types of cells. TMA-DPH and 12-AS fluorescence anisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydrophobic region of plasma membrane in both cell types, but did not influence the fluidity of the outer surface of membrane. We conclude that fibroblasts derived from individuals affected with Down's syndrome are better protected from the damage induced by DNR than normal cells.

Can nurses be employed in 12-hour shift systems?

Nurses are often obliged to work in a 12-hr shift work system. We have decided to check whether such a working system constitutes an excessive load for nurses. On the basis of a questionnaire survey among nurses working in an 8-hr daytime shift system (169 nurses) and in a 12-hr shift (536 nurses), the amount of physical workload, work stress, and after-work activities were compared. Data analysis has shown that a 12-hr shift system is characterized by less significant physical workload but greater mental load. The nurses working in a 2-shift system were more tired after work, but they could spend more time on leisure activities and do housework. The data suggest that there are no significant contraindications for nurses to work in a 2-shift system.

Tempicol-3, a novel piperidine-N-oxide stable radical and antioxidant, with low toxicity acts as apoptosis inducer and cell proliferation modifier of Yoshida Sarcoma cells in vivo.

The novel nitroxyl, Tempicol-3 (nitroxide-N-oxide) was synthesized and its capacity to act as a scavenger of hydroxyl radicals was tested. The concentration-dependent reducibility of this novel compound was also examined and compared with those of previously characterized nitroxides, Tempo and Tempace. The cytotoxicity of Tempicol-3 in vitro was measured by the modified tetrazolium assay (MTT), using, model cells for neoplastic phenotype (mouse NIH 3T3 fibroblast line). The ability of Tempicol-3 to act as an antitumor agent in vivo was also investigated in a pharmacological test, using rats bearing 3-day old Yoshida Sarcoma (promotion phase of the disease). Our results clearly indicated that Tempicol-3 acts as an effective and promising hydroxyl radical scavenger-antioxidant. Structure- and concentration-dependent bioreduction of Tempicol-3 by ascorbic acid may account for some of its biological effects, causing modulation of the antioxidant status of cells. The presence of one nitrone moiety per molecule of Tempicol-3 caused a significant decrease in nitroxide cytotoxicity as compared with Tempo, in vitro. The results clearly confirmed that the toxic effect could result either from the presence or structure of substituent(s) at position 4 of the free radical piperidine moiety. It can be stated that Tempicol-3 is a lowtoxicity nitroxide, which could be effective in providing antioxidative activity. We have also observed that lowtoxic Tempicol-3, at m.e.d. (minimal effective dose) suppressed tumorigenesis, acting as a cell proliferation modifier and apoptosis inducer in vivo. This work provides the base for further investigations on nitroxide-N-oxide derivatives since the serious question remains to be solved as to what is the molecular mechanism of action of the nitroxide-N-oxides.

Effect of zinc on carp (Cyprinus carpio L.) erythrocytes.

The effect of zinc exposure on some properties of the carp erythrocyte membrane was studied in vitro. Red blood cells plasma membranes were separated from other cellular membranes using a combination of differential and density gradient centrifugation. The purity of obtained plasma membrane preparations was determined by measuring the activity of the marker enzymes. Electrophoretic patterns of the main erythrocyte membrane proteins excluded their degradation during the isolation and purification procedure. Carp erythrocyte membranes, obtained from cells previously incubated with increasing ZnSO4 concentrations, were used to elucidate the effect of zinc ions on their physical and biochemical properties. Using fluorescent probes: 12-AS and TMA-DPH, we found that zinc ions reduced the fluidity of the lipid bilayer, both in the middle and near the aqueous interface. Moreover, it was observed that zinc had no significant influence neither on the Na,K-ATPase activity nor on the thiol groups content in the erythrocyte membrane. We also detected that incubation of erythrocytes with zinc lead to the marked decrease of hemolytic resistance of the cells. Our studies demonstrate that zinc at higher concentrations may be toxic to carp erythrocytes causing changes in the membrane fluidity and hemolytic resistance.

Antiproliferative and cytotoxic effects of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with anthracycline(s), anthraquinone and nitroxides action.

Since flavanone oximes derivatives (ethers) have been shown to modulate the growth of Yoshida Sarcoma cells in vivo and to induce apoptosis, the effects of these substances on immortalized cell lines growth were examined. Cell viability and sensitivity to investigated substances was measured by the modified tetrazolium salt (MTT) assay. The antiproliferative effects were expressed as IC50 and IC90, respectively. There were very substantial differences in the dose-dependency of the observed antiproliferative and cytotoxic effects. The structure-activity relationship was evident and revealed that the substitution at B-ring of molecule seems to be an important factor in flavanone oxime (ether) potency. Compared to anticancer drugs (doxorubicin, aclarubicin and mitoxantrone) flavanone oximes displayed cytotoxicity at considerably higher concentrations. The antiproliferative action of the investigated model nitroxides depended on the free radical part of the molecule. N-hydroxy derivative (reduced cation form) did not influence cells proliferation and nor display any cytotoxicity at the applied range about 60 times higher than those of flavanone derivatives. Taken together it seems reasonable to suggest that flavanone oxime(s) (ethers) as compared with antracycline(s), anthraquinone and nitroxides might be especially good candidates for in the future development of new therapeutic techniques.

The effect of daunorubicin and glutaraldehyde treatment on the structure of erythrocyte membrane.

The effect of daunorubicin (DNR) and glutaraldehyde on erythrocyte membrane structure was examined by Electron Spin Resonance spectroscopy. Human erythrocytes were incubated with daunorubicin and then with glutaraldehyde to prevent drug efflux. We have demonstrated that DNR alone caused changes in membrane fluidity mainly in the hydrophobic regions of the lipid bilayer. When DNR-preincubated erythrocytes were treated with glutaraldehyde, the alterations in fluidity were observed in the polar regions as well as in the deeper regions of the cell membrane. The incorporation of drug and glutaraldehyde into human erythrocytes also caused conformational alterations in membrane cytoskeletal proteins and changes in the internal viscosity of the cells. The results suggest that glutaraldehyde in the drug-pretreated erythrocytes may lead to significant perturbations in the organization of the plasma membrane lipids and proteins.

Zinc-induced damage to carp (Cyprinus carpio L.) erythrocytes in vitro.

Fish erythrocytes were used to elucidate the effect of zinc ions on the cell antioxidant defence system. It was detected that an increase of the Zn2+ concentration (0.01-1 mM) leads to a marked decrease (p < 0.05) in the catalase and the glutathione peroxidase activities. We observed a loss of 14-39% activity of glutathione peroxidase, and 16-20% diminution for catalase. No significant changes were found in case of the superoxide dismutase. Incubation of red blood cells with zinc brought about a decrease of the erythrocyte thiol group content. Treatment of carp erythrocytes with zinc ions also resulted in enhanced hemolysis and in the induction of significant (p < 0.001) changes in the intracellular glucose level. The increase of glucose concentration in the erythrocytes was correlated with increased concentration of metal in the incubation medium. It was proposed that Zn could affect transport systems across the red blood cells and therefore increased the permeability of the membranes to small molecules (e.g. hexose), and led to hemolysis. Zinc ions could act as a potential cell toxicant, leading to disturbances in functions of the antioxidant defence system and to alterations in the erythrocyte membrane properties.

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone.

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.

Single-strand breaks and repair in nuclear DNA in the presence of hydralazine assayed by the nucleoid technique.

The nucleoid sedimentation assay was used to study hydralazine-induced DNA structural changes and repair in the fibroblasts cultured in vitro. The drug induced a dose dependent loss in negative DNA supercoiling due to the physical breakage of the DNA. Relaxation of supercoiled DNA resulted in the nucleoids sedimenting with lower velocities than those of undamaged control cultures. Repair incubation of the cells did not cause the restoration of DNA supercoiling to control level. Unsuccessful repair of DNA damaged by hydralazine may result in maintaining the damaged DNA in the cell which could have immunologic consequences.

The effects of imidazoacridinones, new antineoplastic agents, on the human erythrocyte membrane.

In the present study, the interactions of imidazoacridinones, C-1311 and C-1371 with human erythrocyte membranes were examined by electron spin resonance spectroscopy (ESR). It was observed that both compounds C-1311 and C-1371 preferentially bind to proteins producing the conformational changes of erythrocyte membrane skeletal proteins.

The interactions of modified lipoproteins with human erythrocyte membranes.

The interactions of native and modified lipoproteins with erythrocyte membranes were studied via electron paramagnetic resonance (EPR). The in vitro incubation of minimally oxidized and glycated low density lipoprotein (LDL) with erythrocyte membranes of normal subjects induced a slight rigidification of the membrane lipids. The glycated LDL also caused alterations in conformation of normal and diabetic erythrocyte membrane proteins. In contrast both oxidized and glycated high density lipoprotein (HDL) caused no significant changes in the organization of membranes. These, new findings suggest that the modified LDL may influence the structure of erythrocyte membrane components.