Quality and Purity of L929 Cell Cultures: Isoenzyme Profiles As a Sensitive Marker of Cross-Contamination.

Objective: Inappropriate handling of cells can generate modifications in the genomic DNA. The additional risk is cross-contamination. Isoenzyme analysis with gel agarose electrophoresis is a known, fast, and cheap technique for detection of species-specific isoforms of intracellular enzymes. The aim of the experimental work was to check if variations in the cell growth conditions can affect morphology and/or nuclear anomalies including micronuclei (MN) in the L929 cells; and to define how sensitive and selective is the classic gel agarose electrophoresis for analysis of isoforms of the selected enzymes to detect cross-contamination of L929 cultures with HeLa cells or with the related species, such as CHO-K1 cells, in the case of unavailability of the commercial kits. Methods: The experiments were done with use of the National Collection of Type Cultures clone 929 (L929)-mouse fibroblasts from subcutaneous connective tissue; HeLa-human cervix adenocarcinoma; and CHO-K1-epithelial-like hamster ovary cells. Cell morphology was evaluated with a light/fluorescence microscope. MN were determined with the cytokinesis-block micronucleus assay, and the isoenzyme analysis was performed using gel agarose electrophoresis. Results: As shown, the overgrown cultures result in a significant increase of the MN in the L929 cells. The band patterns for lactate dehydrogenate, glucose-6-phosphate dehydrogenase, or malate dehydrogenase allow identification of the single L929, HeLa, or CHO-K1 cell line and to detect the cross-contamination, even up to 0.4%. Conclusions: There can be no exceptions from the recommended cell culture conditions in the passage scheme. The sensitivity of the gel agarose separation depends on the cells and on the type of enzyme tested and seems to be sufficient in a quick screening of the CHO-K1, L929, or HeLa cell cultures through the possible mutual contamination.

Polyurethane Foam Rafts Supported In Vitro Cultures of Rindera graeca Roots for Enhanced Production of Rinderol, Potent Proapoptotic Naphthoquinone Compound.

Unique phytochemical profile of plants belonging to Boraginaceae family provides a prolific resource of lipophilic pigments from the group of naphthoquinone derivatives. To overcome low compound content, the major obstacle of plant-based production, immobilization of Rindera graeca roots in in vitro cultures was implemented for efficient production of rinderol, novel furanonaphthoquinone derivative with anticancer properties. Chromatographic procedures revealed rinderol presence in extracts of all investigated root lines, derived both from root biomass and post-culture medium. Unexpectedly, in the second stage of the experiment, rinderol production was ceased in control, unmodified culture systems. On the contrary, roots immobilized on PUF rafts uniformly and stably produced rinderol, and its highest amount was noted for transformed root lines after 42 days of cultivation (222.98 ± 10.47 µg/flask). PUF occurred to be the main place of compound accumulation. Moreover, investigation of rinderol biological activity revealed its fast-acting cell death induction in HeLa cervical cancer cells at relatively low concentrations. Presented results revealed successful application of R. graeca roots immobilization on PUF rafts for production and in situ product removal of rinderol, novel lipophilic furanonaphthoquinone with suggested proapoptotic activity.

The application of an in vitro micronucleus test in mouse fibroblast L929 cells.

The MNa (in vitro the micronucleus assay) is recommended for studying genotoxicity of chemicals. However, no protocol is currently available for experiments with mouse fibroblast L929 cells. The aim of this study was to improve the scope of CBMNb (cytokinesis-block micronucleus) test. Optimization consisted of: selection of a non-cytotoxic concentration of cytokinesis blocker - cytoBc (cytochalasin B) and type and definition of the positive controls, verification of the efficacy of phenobarbital/5,6-benzoflavone as an S9 enzyme inducer as well as the identification of an optimal staining method. The compounds were tested in three exposure regimens: 6 h exposure with S9 activation followed by a 24 h recovery period, 6 h exposure followed by a 24 h recovery without metabolic activation of S9 and 30 h continuous exposure without S9. Different parameters, such as internal and interlaboratory reproducibility were investigated and criteria for test correctness were proposed. Higher MN rates were achieved using 1 µg/mL cytoBc as a cytokinesis blocker, and MMSd (methyl methanesulfonate), (250 µM), Cole (colchicine), (0.5 µM) and CPf (cyclophosphamide), (30 µM) as positive controls. In regard to the recommended S9 inducer, phenobarbital/5,6-benzoflavone was more effective as Aroclor 1254. Giemsa and acridine orange stains were optimal for the evaluation of MN formation. The protocol described in this study with L929 cells produced the reliable results and is suitable for performing the CBMNb experiments according to the current OECD Guideline #487.

Role of thiamine in Huntington's disease pathogenesis: In vitro studies.

BACKGROUND: Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES: The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS: The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS: Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS: The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Cytotoxicity Evaluation and Crystallochemical Analysis of a Novel and Commercially Available Bone Substitute Material.

BACKGROUND: Alloplastic biomaterials are an alternative for autologous transplants and xenografts in oral surgery and dental implantology. These non-immunogenic and resorbable materials are becoming the basis for complete and predictable guided bone regeneration in many cases. The chemical composition of a great majority of them is based on calcium phosphate salts. In vivo performance is often variable. OBJECTIVES: The objective was to evaluate the biological and chemical properties of an experimental bone substitute material. MATERIAL AND METHODS: The present research focuses on the cytotoxicity comparison and physiochemical characterization of two biomaterials: a novel chitosan/tricalcium phosphate/alginate composite (CH/TCP/Ag) and a commercially available synthetic bone graft made of HA (60%) and ßTCP (40%) (HA/TCP). The materials were evaluated according to PN-EN ISO 10993 Biological evaluation of medical devices i.e. cytotoxicity on mouse fibroblasts (L929) and, in addition, tests on human osteoblasts (hFOB1.19) and human osteosarcoma (MG-63) were conducted. The crystallochemical analysis was performed using the X-ray powder diffraction method. The Bruker-AXS D8 Advance diffractometer (Karlsruhe, Germany) was used to collect diffractograms. RESULTS: The tested materials showed a close resemblance in chemical composition and a considerable differentiation in cytotoxic response. CONCLUSIONS: The novel composite demonstrated a high degree of cytocompatibility, which is promising in future clinical trials.

[The role of thiamine in neurodegenerative diseases]. / Rola tiaminy w chorobach neurodegeneracyjnych.

Vitamin B1 (thiamine) plays an important role in metabolism. It is indispensable for normal growth and development of the organism. Thiamine has a favourable impact on a number of systems, including the digestive, cardiovascular and nervous systems. It also stimulates the brain and improves the psycho-emotional state. Hence it is often called the vitamin of "reassurance of the spirit". Thiamine is a water-soluble vitamin. It can be present in the free form as thiamine or as its phosphate esters: mono-, di- or triphosphate. The main source of thiamine as an exogenous vitamin is certain foodstuffs, but trace amounts can be synthesised by microorganisms of the large intestine. The recommended daily intake of thiamine is about 2.0 mg. Since vitamin B1 has no ability to accumulate in the organism, manifestations of its deficiency begin to appear very quickly. The chronic state of thiamine deficiency, to a large extent, because of its function, contributes to the development of neurodegenerative diseases. It was proved that supporting vitamin B1 therapy not only constitutes neuroprotection but can also have a favourable impact on advanced neurodegenerative diseases. This article presents the current state of knowledge as regards the effects of thiamine exerted through this vitamin in a number of diseases such as Parkinson's disease, Alzheimer's disease, Wernicke's encephalopathy or Wernicke-Korsakoff syndrome and Huntington's disease.

A study of molecular changes relating to energy metabolism and cellular stress in people with Huntington's disease: looking for biomarkers.

Huntington's disease (HD) is a neurodegenerative disorder characterized by a progressive motor and cognitive decline and the development of psychiatric symptoms. The origin of molecular and biochemical disturbances in HD is a mutation in the HTT gene, which is autosomally dominantly inherited. The altered huntingtin protein is ubiquitously expressed in the CNS, as well as in peripheral tissues. In this study we measured the metabolism changes in gene transcription in blood of HD gene carriers (premanifest and manifest combined) versus 28 healthy controls. The comparison revealed statistically significant Global Pattern Recognition Fold Change (FC) for 6 mRNA transcripts, reflecting an increase of: MAOB (FC = 3.07; p = 0.0005) which encodes an outer mitochondrial membrane-bound enzyme called monoamine oxidase type B; TGM2 (FC = 1.8; p = 0.02) encoding a transglutaminase 2 that mediates cellular stress; SLC2A4 (FC = 1.64; p = 0.02) solute carrier family 2 (facilitated glucose transporter) member 4; branched chain ketoacid dehydrogenase kinase (BCKDK) (FC = 1.34; p = 0.02); decrease of LDHA (FC = -1.16; p = 0.03) lactate dehydrogenase A; and brain-derived neurotrophic factor (BDNF) (FC = -2,11; p = 0.03). These distinguished changes coincided with HD progress. The analyses of gene transcription levels in sub-cohorts confirmed these changes and also revealed 28 statistically significant FCs of gene transcripts involved in ATP production and BCAA metabolism.

Modulation of apoptosis protein profiles--role of P-gp in HeLa cells exposed to doxorubicin.

As shown previously doxorubicin (1 µM) plus sulindac (50 µM) reduced the expression of ABCB1 (ATP-binding cassette, sub-family B (MDR/TAP), member 1) mRNA in HeLa cells and this effect was accompanied by increased apoptosis. The aim of this study was to define if the decrease of ABCB1 expression or blocking of P-glycoprotein (P-gp) can affect the expression of the apoptotic genes determined with use of quantitative real time polymerase chain reaction (qRT-PCR). Western blot was used for visualization of chosen pro- and antiapoptotic proteins. Doxorubicin was the main compound which affected the apoptotic genes. The effectiveness of the drugs in reducing of P-gp function has been shown as not being related to the regulation of apoptotic gene transcription. In this experimental scheme, regulation of apoptotic gene transcription depended on the kind of P-gp modulator.

[The role of the proteasome for therapy of incurable diseases]. / Rola proteasomu w terapii chorób nieuleczalnych.

Proteins constitute the basic building elements of living organisms. Proteins have a limited lifetime in a cell. The so called "half-life period" of proteins is diverse and lasts from several minutes to several days. Regulatory proteins appear in a cell for a definite time and are short-lived. The proteins responsible for basic cell functions are stable and long-lived. Once their functions are fulfilled or because of their surfeit or damage, proteins are eliminated by degradation. Transformation processes of proteins are precisely controlled. There is also a strict association between protein metabolism and the energetic state of a cell. The main proteolytic cell systems are lysosomal and proteasome ones. The first (lysosomes) function in a simple and transparent way. The second system (proteasomes) is highly organized; by using ubiquitin it delivers "molecular label" and sends a marked protein for degradation. Efficient degradation of cellular proteins by the UPS route (ubiquitin - proteasome system) is essential for signal transduction, transcription adjustment, response to stress and control of receptors' activity. The dysfunction of the UPS route is crucial in the development of tumors, neurodegenerative diseases and diseases of immunological and infectious origin. Therefore, it is a challenge to elaborate methods of pharmacological intervention within this system involving, for example, the use of specific, low molecular-weight proteasome inhibitors and enzymes catalyzing the ubiquitination process. The article presents a review of advances in the field, including description of the lysosomal protein degradation route, proteasome model, and the phenomenon of protein aggregation. The summary of the experience on applied therapies, which use the processes of protein degradation as a basis, were also presented.

Possible role of HSP60 in synergistic action of anthracyclines and sulindac in HeLa cells.

As was observed in the earlier studies, doxorubicin (DOX) induced apoptosis in HeLa cells and that effect was potentiated significantly by sulindac (SUL). The aim of the current work was to study the effects of DOX and SUL on HSP60, HSF1 and HSP60 expression and the influence of DOX and SUL on HSP60 translocation. Expression of HSP60 and HSF1 was determined with QRT-PCR; the expression and localization of HSP60 were evaluated with Western blot. The 24-h cell cultures were co-incubated with DOX - 1 microM and/or SUL - 50 microM. The significant induction of HSF1 and HSP60 mRNA level was observed after exposure of the cells to DOX 1 microM. SUL 50 microM alone caused moderate increase in mRNA level. The significant decrease in expression of HSF1 and HSP60 was noted after DOX 1 microM and SUL 50 microM simultaneous treatment. HSP60 appeared in the higher levels in cytosol than in mitochondria No intracellular translocation was noted under treatment of the cells to DOX and/or SUL. In conclusion, the effects of HSP60 and HSF1 evoked in the cells depend on the inducer, proapoptotic action of DOX + SUL may correlate to the increased expression of HSF1 and HSP60; HSP60 mRNA level and the regulation of that protein expression depend on the apoptotic inducer; the role of HSP60 in apoptosis expressed in potential shift between mitochondria and cytosol is determined by the apoptotic inducer and the cell type.

Synergistic action of doxorubicin and sulindac in human cervix carcinoma cells - studies on possible mechanisms.

BACKGROUND: Epidemiologic and experimental studies have shown that cyclooxygenase-2 (COX-2) inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs) are effective chemopreventive agents. The mechanisms underlying the antitumor activity of COX-2 inhibitors are thought to involve inhibition of COX-2 enzyme activity and induction of apoptosis. The aim of the current work was to study the mechanisms of synergistic action noted in HeLa cervical carcinoma cells under doxorubicin (DOX) and sulindac (SUL) co-treatment. MATERIAL/METHODS: Cytotoxic activity of the drugs was defined with MTT test, apoptosis was detected with TUNEL test, DOX transmembrane efflux was measured fluorometrically, expression of MDR-1 and MRP-1 was determined with quantitative real time - PCR (QRT-PCR). RESULTS: It was shown that SUL at non-toxic concentrations, 10 and 50 microM, is an effective enhancer of cytotoxic action for DOX in 0.5 and 1 microM, respectively; however, only for SUL concentration equal to 50 microM potentiated apoptosis induced by 1 microM of DOX. Moreover, blocking DOX efflux outside the cells was observed. The QRT - PCR analysis has shown that, when used simultaneously, DOX 1 microM and SUL 50 microM results in decreased mRNA level for MDR-1 and MRP-1. CONCLUSIONS: It is concluded that cytotoxic action of DOX against HeLa cells is enhanced by non-toxic concentrations of SUL. The observed effect is due to quenching of MDR-1 and MRP-1 genes expression, which results in blocking of efflux of DOX outside the cells, which in turn correlates with enhanced apoptotic effects. According to obtained results the mechanisms of potentiating of DOX action by SUL are dose specific.

NFkappaB activation and drug sensitivity in human neoplastic cells treated with anthracyclines.

NFkappaB (nuclear factor kappaB) is a transcription factor controlling, among others, cell proliferation and apoptosis. The potent activators of NFkappaB are anthracyclines which can activate apoptotic processes. As shown by some authors, NFkappaB activated by these drugs well correlated with their cytotoxic activity. The aim of this study was to assess the effects of doxorubicin (DOX) and its analogs (annamycin, WP903) on the NFkappaB activity in human melanoma cells: a sensitive (ME18) and a resistant to DOX (ME18/R) and its possible correlation with cell sensitivity to these drugs. In the studies, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, ELISA test and confocal microscopy were used. As was shown, DOX, 1.7; 8.6 microM, strongly induced NFkappaB in ME18 cells. Annamycin (ANN), 0.3; 3.0 microM and WP903, 3.0 microM induced NFkappaB in ME18/R cells. PDTC (pyrrolidine dithiocarbamate)--NFkappaB inhibitor made ME18/R cells more sensitive to ANN and WP903 but did not affect cytotoxicity of DOX in ME18 cells. These results suggest that the influence of NFkappaB activation on cytotoxicity of anthracyclines is highly drug- and cell-specific.

Effect of structural modification at the 4, 3', and 2' positions of doxorubicin on topoisomerase II poisoning, apoptosis, and cytotoxicity in human melanoma cells.

INTRODUCTION: The mechanism of the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth inhibition seems to be highly specific and dependent on cell type and anthracycline drug. Resistance and the high cardiotoxicity of anthracyclines have stimulated many studies aimed at identifying critical substituents required for optimal activity. Many authors point to the fact that the double-strand breaks, the consequence of the activity of topoisomerase II poisons, and the inability of cells to repair the DNA lesions are the signal for apoptosis. The aim of this study was to define the influence of 4-demetoxy 2'-halogenated analogs with altered basicity at the 3'-position on topoisomerase II and the relationship of that interaction with apoptosis and the cytotoxicity of these novel anthracyclines. Parental human ME18 melanoma cells and the ME18/R subline, obtained experimentally, resistant to doxorubicin (DOX), exposed to 1.7 and 8.6 microM DOX or its analogs, annamycin and WP903 (both 0.3 and 3.0 microM) were studied. MATERIALS AND METHODS: The MTT test was used to assay cytotoxicity. Interaction of the drugs with topoisomerase II and apoptosis were done by Western blot and fluorescence microscopy using Hoechst 33342. RESULTS: The structural changes at positions 4, 2', and 3' can influence topoisomerase II interaction and apoptotic activity, although correlation between these events and cytotoxic consequences has not been proved. CONCLUSIONS: The biological response of the cells to the structurally similar anthracyclines may be variable and probably depends on the cell type which seems to be an additional problem in the multifactorial resistance of tumor cells to anthracyclines.

Relationship between topoisomerase II-DNA cleavable complexes, apoptosis and cytotoxic activity of anthracyclines in human cervix carcinoma cells.

The use of anthracyclines as antitumor drugs dates back to the 1970s, but the mechanism of the cytotoxicity of these compounds has long been a matter of debate. There is increasing evidence indicating that drug-induced cytotoxicity commonly converges on the induction of apoptosis. Many authors point to the fact that double-strand breaks, resulting from stabilization of cleavable complexes, are the signal for the initiation of the apoptotic cascade. In this work, the possible correlation between stabilization of topoisomerase II (topoII)-DNA complexes, apoptosis induction and cytotoxicity was studied. Parental human cervix carcinoma cells, HeLa, and its subline resistant to vinblastine, KB-V1, were exposed to doxorubicin (DOX) and the novel anthracyclines annamycin and WP903, given at the concentrations 0.2 and 2.0 microg/ml (DOX and annamycin) or 0.2 and 1.0 microg/ml (WP903). It was found that annamycin was the strongest topoII poison in HeLa cells at both concentrations used, whereas poly (ADP-ribose) polymerase (PARP) cleavage was observed dose-dependently in KB-V1 cells treated with annamycin or WP903. Simultaneously, apoptosis, observed as cell morphology or phosphatidylserine translocation, was evident in both cell types exposed to the novel anthracyclines, independent of concentration. DOX appeared to be the weakest apoptotic inducer. On the basis of these studies, it can be suggested that topoII poisoning is not the key process leading to apoptosis and seems to be cell specific. PARP cleavage is probably not an evident marker of anthracycline-induced apoptosis which, in turn, does not seem to be the determinant in the cytotoxic action of these compounds. The efficiency of anthracycline antibiotics, interpreted as cytotoxic action, was dependent on cell type.

Constitutive activation of transcription factor NFkappaB as a possible marker of sensitivity of neoplastic cells to anthracyclines.

NFkappaB, a member of the Rel family of transcription factors has been found to be critically important for control of cell proliferation and apoptosis. Although rare, there are systems in which NFkappaB has occurred as pro-apoptotic factor. The potent activators of NFkappaB are anthracycline anticancer drugs which induce the events of apoptosis. These results could point to the pro-apoptotic role of NFkappaB. Recent studies have shown that activation of that transcription factor well correlated with cytotoxic activity of anthracyclines. Potential mechanism through which NFkappaB could play a role in tumorigenesis involves its constitutive activation which was shown in a wide variety of tumour types. The aim of this study was to define the level of activity of NFkappaB in the native human tumour cells differing in sensitivity to anthracycline analogs to study the mechanism responsible for cytotoxic action of these drugs. Constitutive activation of NFkappaB was determined with use of ELISA test and confocal microscopy. As was shown, both methods have occurred as quite comparable. Constitutive activation of NFkappaB was observed in all neoplastic cells tested independently on sensitivity to anthracyclines. However, these results do not exclude the supportive role of NFkappaB in apoptotic activity of these drugs. It is necessary to study the influence of the compounds tested here on NFkappaB activation and on the induction and intensity of apoptotic processes.

Astrocytic response in the rodent model of global cerebral ischemia and during reperfusion.

The present study investigated alterations in astrocytic cells after global cerebral ischemia resulting from cardiac arrest immediately and at several intervals after reperfusion when excessive formation of highly cytotoxic free radicals is known to occur. The cellular fraction of astrocytic origin (glial plasmalemmal vesicles - GPV) was examined by biochemical and immunochemical procedures. A tendency towards an elevation in immunocontent of glial fibrillary acidic protein (GFAP) was noticed after 24 hours whereas a significant increase was observed 7 days post ischemic event. The features of astrocytic stimulation were also observed in electron microscopy studies. An enhanced amount of gliofilaments was noticed in brain sections obtained from rats after 7 days of recovery. Simultaneously, a gradual decrease of total glutathione level, depending on the duration of reperfusion, was observed in brain homogenates and in fractions of astroglial origin. The most considerable reduction was observed on day 1 (52%) and day 7 (65%) after reperfusion in brain homogenates and on day 7 (47%) in GPV fraction. The results indicate an enhanced reactivity of astrocytic cells in ischemic conditions concomitantly with a long lasting decrease of total glutathione. Obviously, the inability of astrocytic glutathione system to detoxify free radicals formed during ischemic/reoxidation conditions may lead to damage to cerebral neurons by oxidative stress.