Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.

Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (α-glyceryl-phosphoryl-choline [αGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 µM (+)lipoic acid or (+/-)lipoic acid and/or 10 mM αGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(+)lipoic acid, (+/-)lipoic acid, or αGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (+)lipoic acid + αGPC or (+/-)lipoic + αGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.

Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design.

The objective of this study was the preparation, physico-chemical characterization and statistical optimization of cationic solid lipid nanoparticles (SLN) prepared by the PIT method as potential carrier for gene therapy, emphasizing the application of factorial design in such a kind of studies. The preliminary screening from a physico-chemical point of view on three cationic lipids (CTAB, DDAB and DOTAP), selected on the basis of their different chemical structure and increasing lipophilicity, allowed us to select SLN with DOTAP, due to its higher zeta potential and smaller particle size. Afterward, a 2(2) full factorial experimental design was developed in order to study the effects of two independent variables (amount of DOTAP and concentration of lipid matrix) and their interaction on mean particle size and zeta potential values. The factorial planning was validated by ANOVA analysis; the correspondence between the predicted values of size and zeta and those measured experimentally confirmed the validity of the design and the equation applied for its resolution. The factorial design showed a significant influence of the independent variables on the selected parameters; in particular, a higher effect of DOTAP was observed on zeta potential value. Different dilutions of the optimized SLN containing 7% w/w of cutina CP and 1% w/w of DOTAP, with size and zeta potential values respectively of 462.9 nm and 50.8 mV, were in vitro examined to evaluate the possible cytotoxicity on two models of cell cultures: human prostate cancer androgen-non-responsive DU-145 cells and primary cultures of rat astrocytes.

Effects of metformin on oxidative stress, adenine nucleotides balance, and glucose-induced insulin release impaired by chronic free fatty acids exposure in rat pancreatic islets.

BACKGROUND: In rat pancreatic islets, chronic exposure to high free fatty acid (FFA) levels impairs insulin secretion and ß cell mass. The mechanisms underlying this defect are not completely understood. Since islets have intrinsically low anti-oxidant enzyme defense, oxidative stress might be responsible for ß cell damage. AIM: In this study, we investigated if FFA could induce oxidative stress in rat pancreatic islets and if metformin might reverse adverse effects. MATERIAL AND METHODS: We cultured rat pancreatic islets in the presence or absence of FFA (oleate/palmitate 2:1, 2 mM) for 72 h. In some experiments, we used metformin (2.5 µg/ml) during the last 24 h. RESULTS: In our model, glucosestimu lated insulin release was markedly reduced (p<0.005) after chronic FFA exposure, and the ATP/ADP ratio was altered (p<0.05). We observed a significant increase of reactive oxygen species (ROS) (p<0.001), malondialdehyde a lipid peroxidation product (p<0.01) and nitric oxide (NO) levels in the culture media (p<0.001). Inducible NO synthase (iNOS) and heat shock protein-70 (HSP-70) protein expression were also increased (p<0.001 and p<0.01, respectively). When metformin was present during the last 24 h of culture, insulin secretion was restored, and the ATP/ADP ratio was normalized. ROS production, NO production, lipid peroxidation, iNOS and HSP-70 protein expression levels had decreased. CONCLUSIONS: These data indicate that, in rat pancreatic islets, chronic exposure to high FFA induces oxidative stress and that metformin, by reducing this effect, may have a direct beneficial effect on insulin secretion impaired by lipotoxicity.

Alpha-lipoic acid modulates GFAP, vimentin, nestin, cyclin D1 and MAP-kinase expression in astroglial cell cultures.

In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 µM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.

Cell death in human acute myelogenous leukemic cells induced by pyrrolidinedithiocarbamate.

Pyrrolidinedithiocarbamate (PDTC) is a metal chelating compound, which exerts both pro-apoptotic effect and pro-oxidant activity on many cells. Our objective was to investigate whether PDTC was able to interfere with apoptotic process in leukemic and normal bone marrow CD34+ cells. Since hematopoietic growth factors stimulate growth and differentiation and prevent apoptosis, we therefore studied the effect of growth factors pretreatment, such as interleukin-3 and granulocyte-macrophage colony stimulating factor, in human myeloid CD34+ cells to evaluate whether they protect the cells from the apoptotic action of PDTC. We revealed that PDTC exerted an apoptotic effect in leukemic CD34+ cells. This effect was dependent on the ability of this compound to generate the oxidation of cellular glutathion to its disulphide and consequently to induce an intracellular oxidative stress. Hematopoietic growth factors did not protect cells from apoptosis induced by previous treatment with PDTC. The ability of PDTC to induce apoptosis was restricted to acute myelogenous leukaemia CD34+ cells, since normal CD34+cells were insensitive to the pro-oxidant effect of PDTC. These findings imply that normal cells are equipped with mechanisms by which they respond differently to PDTC effects with respect to leukemic cells.

Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts.

Perturbation of oxidant/antioxidant cellular balance, induced by cellular metabolism and by exogenous sources, causes deleterious effects to proteins, lipids, and nucleic acids, leading to a condition named "oxidative stress" that is involved in several diseases, such as cancer, ischemia-reperfusion injury, and neurodegenerative disorders. Among the exogenous agents, both H(2)O(2) and hyperthermia have been implicated in oxidative stress promotion linked with the activation of apoptotic or necrotic mechanisms of cell death. The goal of this work was to better understand the involvement of some stress-related proteins in adaptive responses mounted by human fibroblasts versus the oxidative stress differently induced by 42 degrees C hyperthermia or H(2)O(2.) The research was developed, switching off inducible nitric oxide synthase (iNOS) expression through antisense oligonucleotide transfection by studying the possible coregulation in the expression of HSP32 (also named HO-1), HSP70, and iNOS and their involvement in the induction of DNA damage. Several biochemical parameters, such as cell viability (MTT assay), cell membrane integrity (lactate dehydrogenase release), reactive oxygen species formation, glutathione levels, immunocytochemistry analysis of iNOS, HSP70, and HO-1 levels, genomic DNA fragmentation (HALO/COMET assay), and transmembrane mitochondrial potential (deltaPsi) were examined. Cells were collected immediately at the end of the stress-inducing treatment. The results, confirming the pleiotropic function of i-NOS, indicate that: (i). HO-1/HSP32, HSP70, and iNOS are finely tuned in their expression to contribute all together, in human fibroblasts, in ameliorating the resistance to oxidative stress damage; (ii). ROS exposure, at least in hyperthermia, in human fibroblasts contributes to growth arrest more than to apoptosis activation; and (iii). mitochondrial dysfunction, in presence of iNOS inhibition seems to be clearly involved in apoptotic cell death of human fibroblasts after H(2)O(2) treatment, but not after hyperthermia.

Red wine micronutrients as protective agents in Alzheimer-like induced insult.

Plant extract micronutrients are commonly added to diets for health and prevention of degenerative disease. However, there are barriers to the introduction of these products as antioxidant therapies in counteracting chronic human diseases, probably because the molecular bases of their therapeutic potential are poorly clarified. The present study was designed to evaluate the possible protective effect of combined micronutrients present in black grape skin on toxicity induced by 25-35 beta-amyloid peptid or by serum of Alzheimer's disease patients, in human umbilical vein endothelial cells (HUVECs). The hypothesis was tested by examining the results of lactic dehydrogenase (LDH) release to estimate cytoplasmic membrane breakdown; activity of mitochondrial complexes, reactive oxygen species (ROS) production and malonyl dialdehyde (MDA) levels as markers of oxidative stress induction and COMET assay to evaluate DNA fragmentation. The results demonstrate that black grape skin extract reduces the ROS production, protects the cellular membrane from oxidative damage, and consequently prevents DNA fragmentation. The experimental results suggest that this natural compound may be used to ameliorate the progression of pathology in AD disease therapy.

DNA damage in astrocytes exposed to fumonisin B1.

Fumonisins are a group of toxic metabolites mainly produced by Fusarium moniliforme and Fusarium proliferatum, fungi that commonly occur on corn throughout the world. Fumonisin B1 (FB1), structurally resembling sphingoid bases, is an inhibitor of ceramide synthase, a key enzyme involved in de novo sphingolipid biosynthesis and in the reacylation of free sphingoid bases derived from sphingolipid turnover. This inhibitory effect leads to accumulation of free sphinganine (SA) and sphingosine (SO), inducing cell death. However, little is known on the down stream effectors activated by these sphingolipids in the cell death signaling pathway. We exposed rat astrocytes to FB1 with the aim of evaluating the involvement of oxygen free radicals and of some other biochemical pathways such as caspase-3 activity and DNA damage. Our results indicate that FB1 treatment (48, 72 h and 6 days in vitro, DIV, and 10, 50, 100 microM) does not affect cell viability. Conversely, after 72 h of treatment, FB1 (50 and 100 microM) induced DNA damage and an enhancement of caspase-3 activity compared to controls. In addition, FB1 increased the expression of HSP70 at 10 and 50 microM at 48, 72 h, and 6 DIV of treatment. We conclude that DNA damage of apoptotic type in rat astrocytes is caused by FB1 and that the genotoxic potential of FB1 has probably been underestimated and should be reconsidered.

DNA damage in human fibroblasts exposed to fumonisin B(1).

Fumonisins are mycotoxins produced by several Fusarium species (Fusarium verticilloides and F. proliferatum) that infest corn and other cereals. Fumonisin B(1) (FB(1)), structurally resembling sphingoid bases, is an inhibitor of ceramide synthetase, a key enzyme involved in de novo sphingolipid biosynthesis and in the reacylation of free sphingoid bases derived from sphingolipid turnover. This inhibitory effect leads to accumulation of free sphinganine and sphingosine and subsequent induction of cell death. However, the downstream effectors activated by these sphingolipids in the cell death-signalling pathway are little known. The aim of this study was to evaluate, in FB(1)-exposed human fibroblasts, the involvement of oxygen free radicals and of some other biochemical pathways, caspase-3 activity, poly(ADP-ribose)polymerase (PARP) cleavage and DNA damage evaluated by comet assay. Our results indicate that FB(1) treatment (48, 72 h and 10, 50, 100 microM) does not affect cellular viability. Conversely, after 72 h of treatment, FB(1) (50 and 100 microM) induced DNA damage, an enhancement of caspase-3-activity and cleavage of PARP compared to controls. In addition, FB(1) increased the expression of HSP70 in a concentration and time-dependent manner. Our results indicate that DNA damage of apoptotic type in human fibroblasts is caused by exposure to FB(1) at high concentrations and for a prolonged time and that the genotoxic potential of FB(1) has probably been underestimated and should be reconsidered.

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70.

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress-induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions.

Effects of Gabapentin and Topiramate in primary rat astrocyte cultures.

We studied in vitro the effects of anticonvulsant drugs Gabapentin and Topiramate on the production of reactive oxygen species and nitric oxide (NO), the activity of glutamine synthetase (GS), and cell viability in primary cultures of rat cortical astrocytes which are intimately involved in the normal functioning of neurons. We investigated the effects of these drugs at concentrations within the therapeutic range (1, 10 and 50 microg/ml). We observed that, in cultured astrocytes, Gabapentin induced a weak increase in the biosynthesis of NO, a mild decrease in GS activity and cell viability, and minor induction of a stress condition. Topiramate was observed to induce even greater stressor effects on these cells.

Tiagabine treatment and DNA damage in rat astrocytes: an in vitro study by comet assay.

We studied in vitro the effects of Tiagabine on genomic DNA of cortical rat astrocytes. To evaluate DNA damage, we used a relatively simple technique called Single Cell Gel Electrophoresis or Comet assay. Tiagabine was dissolved in culture medium and added at concentration of 1, 10, 20 and 50 microg/ml on 12-day old cultured astrocytes. In presence of 1 and 10 microg/ml of Tiagabine, no DNA damage was observed after 48 h of treatment. A moderate DNA damage was instead observed for cells exposed to 20 microg/ml of antiepileptic drug. Finally, DNA fragmentation was more evident after treatment with 50 microg/ml of Tiagabine. We conclude that Tiagabine, at the usual recommended doses, does not appear to influence negatively the cortical rat astrocytes, inducing DNA fragmentation only at very high concentrations.

ET-18-OCH(3)-induced cytotoxicity and DNA damage in rat astrocytes.

The ether lipid 1-octadecyl-2-methyl-rac-glicero-3-phosphocholine (ET-18-OCH(3)) is known to be selectively cytotoxic toward several types of tumor cells, in which it seems to activate a process of apoptotic cell death. Moreover, the drug has been demonstrated to be active in normal cells too, particularly in rat astrocytes. In these cells at low dosage (from 1 to 6 microg/ml of medium) ET-18-OCH(3) stimulates maturation and protective responses, whereas at increasing dosages (from 8 to 20 microg/ml) it shows cytotoxic effects. The present study demonstrates that when ET-18-OCH(3) is added to astrocytes, it activates, in a time- and concentration-dependent manner, an oxidative process by increasing both the generation of reactive oxygen species (ROS), including nitric oxide, and lipid peroxidation. When there is a high ET-18-OCH(3) concentration or the time of treatment is prolonged, the increased oxidative condition seems to trigger DNA fragmentation (monitored by COMET assay) as well as loss in cell viability. These cytotoxic effects indicate that ROS may be considered, in our experimental model, as executioners of a program of cell death. In addition, ET-18-OCH(3) being a promising molecule in antitumor therapy, our data, while reinforcing the importance of monitoring the therapeutic drug dosage employed, also suggest that it may be useful to associate some antioxidants with antitumor treatments.

Biological effects of tiagabine on primary cortical astrocyte cultures of rat.

Biological effects of tiagabine, a new antiepileptic drug, were analyzed on cultures of rat's cortical astrocytes. Tiagabine was added to the cultures at concentrations of 1 and 10 microg/ml, correspondent to therapeutic range; cell viability (tetrazolium salt assay and lactic dehydrogenase release), maturation and differentiation (glutamine synthetase activity) and presence of stress conditions (reactive oxygen species formation, inducible nitric oxide synthetase expression and 70 kDa heath shock protein production) were tested. Our results indicate that the addition of Tiagabine to primary astrocytes not only did not change significantly the examined metabolic activities but also seems to exert a protective action against oxidative stress. Thus, our data reinforce the idea that Tiagabine may be considered an effective promising drug in the treatment of epilepsy.

Molecular mechanisms of resistance in Pseudomonas aeruginosa to fluoroquinolones.

Pseudomonas aeruginosa is important in the field of infectious disease especially with respect to its role in nosocomial infections. Infections with P. aeruginosa may be a problem as the organism has intrinsic resistance to several antibiotics and a capability in acquiring resistance during antibiotic therapy. Fluoroquinolones are sometimes used during antibiotic therapy of P. aeruginosa infections even though resistance to fluoroquinolones may develop. Six strains of P. aeruginosa were studied in an attempt to elucidate the mechanisms of resistance to fluoroquinolones. These included the electrophoresis patterns of the outer membrane proteins (OMPs), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) analyses. A method is described that improved the clarity of the OMP gels. Resistance in these P. aeruginosa strains could depend not only on DNA-gyrase modifications but also on membranes alterations and on the presence (qualitative and quantitative) of the efflux pump formed by three subunits.

Deletions in the mitochondrial DNA and decrease in the oxidative phosphorylation activity of children with Fanconi syndrome secondary to antiblastic therapy.

The aim of this study is to verify whether there are deletions in mitochondrial DNA (mtDNA) and disorders in oxidative phosphorylation (Ox-phos) complexes in the pathogenesis of secondary Fanconi syndrome (FS). We studied 18 children with tumors who were previously treated with chemotherapy and were off therapy for at least 1 year. All the children had normal renal function at diagnosis. Only 4 children received ifosfamide (IFO) and platinum compounds. We evaluated renal function, Ox-phos activity measured on platelets, and mtDNA extracted from platelets for all patients. Only 2 patients, both treated with IFO and carboplatinum (CARBO) for Wilms' tumor and germ-cell tumor, respectively, developed FS 1 and 3 years after termination of therapy. They had decreased activities of Ox-phos that were statistically significant only for nicotinamide adenine dinucleotide (NAD)-reduced cytochrome-c reductase and cytochrome-c oxidase and specific and unidentified deletions in mtDNA that were not maternally inherited. Our data suggest that treatment with IFO and CARBO might be responsible for deletions in mtDNA, decreased activity of Ox-phos, and impaired rates of transport of D-glucose, phosphate, and amino acids.

Stress proteins and SH-groups in oxidant-induced cellular injury after chronic ethanol administration in rat.

It is generally agreed that lipid peroxides play an important role in the pathogenesis of ethanol-induced cellular injury and that free sulfhydryl groups are vital in cellular defense against endogenous or exogenous oxidants. It has been observed that oxidative stress induces the synthesis of the 70-kDa family of heat-shock proteins (HSPs). Induction of HSPs represents an essential and highly conserved cellular response to a variety of stressful stimuli. In the present study we measured in various brain areas and in liver the intracellular levels of HSP70 proteins, sulfhydryl groups and the antioxidant enzyme status after chronic administration of mild intoxicating doses of ethanol to rats. Expression of HSP70 in response to alcohol administration was particularly high in the hippocampus and striatum. In these brain areas, the increase in HSP70 protein levels occurred in absence of significant changes of antioxidant enzyme activities and was correlated with a marked depletion of intracellular bound thiols and with a decreased susceptibility to lipid peroxidation. Lower levels of HSP70 induction were found in cortex and cerebellum and were associated to decreases in SOD and CAT enzyme activities, with a lower depletion of protein bound thiols and with an increased susceptibility to lipid peroxidation. This study agrees with our previous results performed on acute alcohol intoxication and supports the hypothesis that HSP70 induction protects the different brain areas against oxidative stress.

Glutamine synthetase activity and HSP70 levels in cultured rat astrocytes: effect of 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine.

The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a membrane interactive drug selectively cytotoxic toward neoplastic cells compared to normal cells. It induces apoptosis in human leukemic HL-60, T-lymphoid and in U937 myeloid cell lines and stimulates NO biosynthesis in cultured rat astrocytes. We have found a double action of ET-18-OCH3 in astrocytes which, at low doses, promotes a moderate induction of heat shock proteins of 70 kDa (HSP70) and the increase of glutamine synthetase (GS) activity. Conversely, at high doses, the drug shows toxic effects on astrocytes inducing decrease in GS activity, low molecular weight DNA formation, and release of lactic dehydrogenase (LDH) in the culture medium. Its analog compound platelet-activating factor (PAF) shares some of these biological aspects.

Nuclear DNA strand breaks during ethanol-induced oxidative stress in rat brain.

Free radical-mediated oxidative damage has been implicated in the pathophysiological mechanisms of apoptosis. In this study we report that statistically significant strand breaks were induced primarily in the hippocampus and cerebellum during chronic, and not acute, ethanol treatment. Damage to DNA observed in hippocampus and cerebellum was also correlated with significant modification in the activities of mitochondrial respiratory complexes I and IV and with a significant increase in lipid peroxidation products. This finding lends support to the fact that hippocampus and cerebellum are brain areas particularly vulnerable to redox changes induced by alcohol intoxication, suggesting lower threshold levels of ethanol tolerance.

Effects of ether lipid 1-O-octadecyl-2-methoxy-rac-glycero- 3-phosphocholine and its analogs PAF and CPAF on the release of nitric oxide in primary cultures of rat astrocytes.

Ether lipid 1-O-octadecyl-2-O-methoxy-rac-glicero-3-phosphocholine (ET-18-OCH3) is an immunomodulator with antineoplastic activity. Its analog compounds PAF and CPAF share some of its biological effects. In our experiments, even very small amounts of ET-18-OCH3 released a remarkable quantity of nitric oxide (NO) from rat astrocytes cultured in vitro. The NO biosynthesis was inhibited by pretreatment with the antagonist BN 50730. The effect of ET-18-OCH3 was greater than that of the LPS inducer. PAF did not produce NO, even at high doses, while the nonmetabolizable CPAF only induced a significant release of NO from 12 micrograms/ml onwards. These results demonstrate that ET-18-OCH3 is functionally active also in astrocyte cultures. Stimulation of NO biosynthesis is of a great value on account of its the known effect as a neurotransmitter, potentiator of immune defences and possible modulator of cerebral circulation.