Metabolic Biomarkers Differentiate a Surgical Intervertebral Disc from a Nonsurgical Intervertebral Disc.

BACKGROUND: Degeneration of the intervertebral disc (IVD) is caused by disturbances in metabolic processes, which lead to structural disorders. The aim of this report is to analyze metabolic disorders in the degeneration process by comparing control discs with degenerated discs. In our research on the nucleus pulposus (NP), we used NMR spectroscopy of extracts of hydrophilic and hydrophobic compounds of the tissue. METHODS: Nuclear magnetic resonance (NMR) spectroscopy allows the study of biochemistry and cellular metabolism in vitro. Hydrophilic and hydrophobic compounds were extracted from the NP of the intervertebral disc. In the NMR spectra, metabolites were identified and quantitatively analyzed. The results of our research indicate disturbances in the biosynthesis and metabolism of cholesterol, the biosynthesis and degradation of various fatty acid groups, ketone bodies, or lysine, and the metabolism of glycerophospholipids, purines, glycine, inositol, galactose, alanine, glutamate, and pyruvate in the biosynthesis of valine and isoleucine, leucine. All these disorders indicate pathomechanisms related to oxidative stress, energy, neurotransmission disturbances, and disturbances in the structure and functioning of cell membranes, inflammation, or chronic pain generators. CONCLUSIONS: NMR spectroscopy allows the identification of metabolites differentiating surgical from nonsurgical discs. These data may provide guidance in in vivo MRS studies in assessing the severity of lesions of the disc.

Serum metabolic profiles and metal levels of patients with multiple sclerosis and patients with neuromyelitis optica spectrum disorders - NMR spectroscopy and ICP-MS studies.

BACKGROUND AND OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a disease misdiagnosed with multiple sclerosis (MS). We hypothesized that the serum metabolic profile could be helpful in the differentiation of both diseases in an early stage. METHODS: We included controls, patients with MS diagnosed according to the McDonald criteria of 2010, and patients with NMOSD diagnosed according to the criteria from 2015. Blood samples were collected on clots from all participants after overnight overfasting. We obtained metabolic profiles using proton magnetic resonance spectroscopy (1HNMR) of serum hydrophilic and hydrophobic compounds. Serum metal levels were measured using isotope-specific detection mass spectrometry (ICP-MS). For statistical analyzes, we used ANOVA tests and multivariate analysis (MVA) - orthogonal partial least square discriminant analysis (OPLS-DA). RESULTS: We analyzed metabolite levels in patient groups compared to controls. We observed significantly different levels of ten metabolite signals in patients with MS vs controls and eighteen metabolite signals in patients with NMSOD vs controls. We observed significantly different levels of five signals in patients with MS vs NMOSD. In the MVA analysis of patient groups, we indicated compounds that most differentiated the groups. All of these compounds are involved in cycles connected to the inflammation process and/or oxidative stress. The results of metallomics studies confirmed metal participation in these processes. DISCUSSION: It is possible to distinguish patients with MS and NMOSD from controls using ANOVA and MVA tests. The chosen metabolite profile analyzes might possibly be helpful in distinguishing the two diseases from each other in some seronegative and radiologically negative cases.

Zinc and Copper Brain Levels and Expression of Neurotransmitter Receptors in Two Rat ASD Models.

Zinc and copper are important trace elements necessary for the proper functioning of neurons. Impaired zinc and/or copper metabolism and signaling are implicated in many brain diseases, including autism (ASD). In our studies, autistic-like behavior in rat offsprings was induced by application to pregnant mothers valproic acid or thalidomide. Zinc and copper contents were measured in serum and brain structures: hippocampus, cerebral cortex, and cerebellum. Our research shows no interconnections in the particular metal concentrations measured in autistic animal brains and their sera. Based on patient researches, we studied 26 genes belonging to disturbed neurotransmitter pathways. In the same brain regions, we examined the expression of genes encoding proteins of cholinergic, adrenergic, serotonin, and dopamine receptors. In both rats' ASD models, 17 out of the tested gene expression were decreased. In the cerebellum and cerebral cortex, expression of genes encoding cholinergic, adrenergic, and dopaminergic receptors decreased, whereas in the hippocampus only expression of serotoninergic receptors genes was downregulated. The changes in metals content observed in the rat brain can be secondary phenomena, perhaps elements of mechanisms that compensate for neurotransmission dysfunctions.

Peptide Dendrimers with Non-Symmetric Bola Structure Exert Long Term Effect on Glioblastoma and Neuroblastoma Cell Lines.

BACKGROUND: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system (CNS). Neuroblastoma (NB) is one of the most common cancers of childhood derived from the neural crest cells. The survival rate for patients with GBM and high-risk NB is poor; therefore, novel therapeutic approaches are needed. Increasing evidence suggests a dual role of redox-active compounds in both tumorigenesis and cancer treatment. Therefore, in this study, polyfunctional peptide-based dendrimeric molecules of the bola structure carrying residues with antiproliferative potential on one side and the antioxidant residues on the other side were designed. METHODS: We synthesized non-symmetric bola dendrimers and assessed their radical scavenging potency as well as redox capability. The influence of dendrimers on viability of rat primary cerebellar neurons (CGC) and normal human astrocytes (NHA) was determined by propidium iodide staining and cell counting. Cytotoxicity against human GBM cell lines, T98G and LN229, and NB cell line SH-SY5Y was assessed by cell counting and colony forming assay. RESULTS: Testing of CGC and NHA viability allowed to establish a range of optimal dendrimers structure and concentration for further evaluation of their impact on two human GBM and one human NB cell lines. According to ABTS, DPPH, FRAP, and CUPRAC antioxidant tests, the most toxic for normal cells were dendrimers with high charge and an excess of antioxidant residues (Trp and PABA) on both sides of the bola structure. At 5 µM concentration, most of the tested dendrimers neither reduced rat CGC viability below 50-40%, nor harmed human neurons (NHA). The same dose of compounds 16 or 22, after 30 min treatment decreased the number of SH-SY5Y and LN229 cells, but did not affect the number of T98G cells 48 h post treatment. However, either compound significantly reduced the number of colonies formed by SH-SY5Y, LN229, and T98G cells measured 14 days after treatment. CONCLUSIONS: Peptide dendrimers with non-symmetric bola structure are excellent scaffolds for design of molecules with pro/antioxidant functionality. Design of molecules with an excess of positive charges and antioxidant residues rendered molecules with high neurotoxicity. Single, 30 min exposition of the GBM and NB cell lines to the selected bola dendrimers significantly suppressed their clonogenic potential.

The effects of exercise training on lipid profile in patients with sarcoidosis.

This study aimed to determine the use of lipid profiling to assess the effects of moderate intensity exercise training (ET) on patients with sarcoidosis. Fourteen patients with sarcoidosis (mean age, 46.0 ± 9.6 years) were examined before and after 3-week of ET programme in hospital settings. Symptoms (fatigue: FAS, dyspnoea: MRC), lung function tests and physical function tests (6 MWT, muscle force) were measured before and after ET. Proton nuclear magnetic resonance (NMR) spectroscopy combined with orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to determine lipid profile before and after ET. Twenty-five NMR signals from lipid compounds were selected for further analysis as well as serum lipid and inflammatory markers. Three weeks of ET results in improvement of symptoms (FAS: 27.5 vs. 21.0; p < 0.001, MRC: 0.86 vs. 0.14; p = 0.002) and physical function (6MWT: 508.43 vs. 547.29; p = 0.039). OPLS-DA analysis of the lipid profiles of patients with sarcoidosis revealed differences among the samples before and after ET, including decreases in fatty acids (p < 0.017), triglycerides (p < 0.022) and total cholesterol (p < 0.020). Other changes included shifts in fatty acids oxidation products and triacylglycerol esters. A short-time, in-hospital exercise training benefits patients with sarcoidosis by enhancing their physical function. Additionally, positive effect on lipid profile was observed also in this study. It is suggested that lipid profiling could become a new prognostic method to assess effects of pulmonary rehabilitation in patients with sarcoidosis.

Changes in the metabolic profiles of the serum and putamen in Parkinson's disease patients - In vitro and in vivo NMR spectroscopy studies.

The aim of this study was to investigate the relationship between serum metabolomic biomarkers and brain in vivo magnetic resonance spectroscopy (MRS) biomarkers in patients with Parkinson's disease (PD) as well as to investigate compound concentration changes by comparing the results with healthy control subjects. Univariate statistical analysis of the serum showed significant differences in the levels of phenylalanine, tyrosine, lysine, glutamine, glutamate, acetone, acetate, 3-hydroxybutyrate, and 1-monoacylglycerol (1-MAG) between the PD patient group and the control group. Orthogonal partial least squares discriminant analysis showed significantly different compound concentrations of acetate, 3-hydroxybutyrate, glutamine, tyrosine, 1-MAG and testosterone. In vivo MRS of the putamen showed significantly higher concentrations of glutamine/glutamate complex and glutamine in patients with PD in comparison to control subjects. Following disrupted metabolic pathways in patients with PD were identified: dopamine synthesis, steroid hormone biosynthesis, fatty acid biosynthesis, the synthesis and degradation of ketone bodies, the metabolism of pyruvate, arginine, proline, alanine, aspartate, glutamate, tyrosine and phenylalanine. The obtained results may indicate changes in neurotransmission, disturbances in energy production and an altered cell membrane structure.

Changes in the Expression of SNAP-25 Protein in the Brain of Juvenile Rats in Two Models of Autism.

The results of genetic studies suggest a possible role for SNAP-25 polymorphism in the development of autism spectrum disorders (ASDs); however, there are no data available on whether changes in SNAP-25 expression also affect animals in rodent models of ASD. The aim of the present study was to explore this issue. The studies included 1-month-old rats representing valproic acid (VPA)- and thalidomide (THAL)-induced models of autism. Their mothers received single doses of VPA (800 mg/kg) or THAL (500 mg/kg) per os on the 11th day of gestation. SNAP-25 protein content in the cerebellum, hippocampus, and frontal lobe was determined using Western blotting, while changes of mRNA levels of Snap25 gene were determined using real-time polymerase chain reaction. Compared to controls, SNAP-25 content was decreased by approximately 35% in all brain structures tested, in both males and females, exclusively in the VPA group. In contrast to this, Snap25 expression, studied in males, was increased in the hippocampus and cerebellum in both, VPA- and THAL-treated rats. We discuss the compliance of these results with the hypothesized role of SNAP-25 in the pathophysiology of ASD and the adequacy of the experimental models used.

Altered expression of glutamatergic and GABAergic genes in the valproic acid-induced rat model of autism: A screening test.

Autism spectrum disorders (ASD) include neurodevelopmental disorders in which behavioral deficits can result from neuronal imbalance of excitation to inhibition (E/I) in the brain. Here we used RT-qPCR to screen for the expression of 99 genes associated with excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission in the cerebral cortex, hippocampus and cerebellum of rats in an established VPA model of ASD. The largest changes in the expression of glutamatergic genes were found in the cerebral cortex, where 12 genes including these encoding some of the subunits of the ionotropic glutamate receptors, were upregulated, while 2 genes were downregulated. The expression of genes encoding the presynaptic glutamatergic proteins vGluT1 and mGluR7 and PKA, involved in downstream glutamatergic signaling, was elevated more than 100-fold. Changes in GABAergic gene expression were found in the cortex, cerebellum and hippocampus; 3 genes were upregulated, and 3 were downregulated. In conclusion, these results revealed that, in the ASD model, several glutamatergic genes in the rat cerebral cortex were upregulated, which contrasts with small and balanced changes in the expression of GABAergic genes. The VPA rat model, useful in studying the molecular basis of ASD, may be suitable for testing experimental therapies in these disabilities.

Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers.

BACKGROUND: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1'-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20⁻100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2⁻20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug⁻target interactions.

Tetrabromobisphenol A-induced depolarization of rat cerebellar granule cells: ex vivo and in vitro studies.

The brominated flame retardant tetrabromobisphenol A (TBBPA) is toxic to cultured brain neurons, and glutamate receptors partially mediate this effect; consequently, the depolarizing effect of TBBPA on neurons is to be expected, but it is yet to be actually demonstrated. The aim of this study was to detect TBBPA-evoked depolarization and identify the underlying mechanisms. The plasma membrane potential of rat cerebellar granule cells (CGC) in cerebellar slices or in primary cultures was measured using whole-cell current clamp recordings, or the fluorescent probe oxonol VI, respectively. The contribution of NMDA and AMPA receptors, voltage-gated sodium channels and intracellular calcium mobilization was tested using their selective antagonists or inhibitors. Direct interactions of TBBPA with NMDARs were tested by measuring the specific binding of radiolabeled NMDAR ligands to isolated rat cortical membrane fraction. TBBPA (25 µM) strongly depolarized CGC in cerebellar slices, and at ≥ 7.5 µM concentration-dependently depolarized primary CGC cultures. Depolarization of the primary CGC by 25 µM TBBPA was partly reduced when MK-801 was applied alone or in combination with either TTX or CNQX, or where bastadin 12 was applied in combination with ryanodine, whereas depolarization was completely prevented when MK-801, CNQX and TTX where combined. TBBPA had no effect on the specific binding of NMDAR radio-ligands to isolated cortical membranes. These results demonstrate the depolarizing effect of TBBPA on CGC, which is mainly mediated by ionotropic glutamate receptors, while voltage-gated sodium channels are also involved. We found no evidence for the direct activation of NMDARs by TBBPA.

Glutamate, Glutamine and GABA Levels in Rat Brain Measured Using MRS, HPLC and NMR Methods in Study of Two Models of Autism.

The disorders of the glutamatergic neurotransmission have been associated with pathogenesis of autism. In this study we evaluated the impact of the in vivo and ex vivo test methodology on measurements of levels of neurotransmitter amino acids in hippocampus of rats for valproic acid- (VPA) and thalidomide- (THAL) induced models of autism. The main goal was to compare the changes in concentrations of glutamate (Glu), glutamine (Gln) and GABA between both autistic groups and the control, measured in vivo and ex vivo in homogenates. The rat pups underwent three in vivo tests: ultrasonic vocalization (USV), magnetic resonance spectroscopy (MRS) and unilateral microdialysis of the hippocampus. Analyses of homogenates of rat hippocampus were performed using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. For the statistical analysis, we performed univariate and multivariate tests. USV test, which is considered in rodents as an indicator of pathology similar to autism, showed decreased USV in VPA and THAL groups. In vivo MRS studies demonstrated increases of Glu content in male rat's hippocampus in VPA and THAL groups, while the microdialysis, which allows examination of the contents in the extracellular space, detected decreases in the basal level of Gln concentrations in VPA and THAL groups. Ex vivo HPLC studies showed that levels of Glu, Gln and GABA significantly increased in male rat's hippocampus in the VPA and THAL groups, while NMR studies showed increased levels of Gln and GABA in the VPA group. Collectively, these results are consistent with the hypothesis suggesting the role of the glutamatergic disturbances on the pathogenesis of autism. For all methods used, the values of measured changes were in the same direction. The orthogonal partial least square discriminant analysis confirmed that both animal models of autism tested here can be used to trace neurochemical changes in the brain.

Serum lipidomics in diagnostics of sarcoidosis.

The aim of this study was to determine the use of the lipid profile of patients with sarcoidosis and compare it with healthy subjects. We assume that lipid profile of serum in sarcoidosis differs from the lipid profile of control subjects. Serum was collected from 14 patients with II stage of sarcoidosis and 14 control subjects (healthy volunteers). Proton NMR spectroscopy combined with discriminant analyses, OPLS-DA (orthogonal partial least squares projections to latent structures discriminant analysis), was used. Thirty four NMR signals of lipid compounds were selected. OPLS-DA model consisted of three components and very good explain the data and also predict the data. Discriminant analysis correctly classified patients according to their groups for 92.9% of sarcoidose and 100% of control. From multivariate discriminant analysis we obtain a list of potentialbiomarkers which are statistically significant and which separate one class from another. These biomarkers are statistically significant, but not necessarily biochemically significant. They may have biochemical significance and they may be the biomarkers we are interested in, however, this must be established through extensive testing. Presented method allows distinguishing between healthy subject and sarcoidosis patients. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 150-153).

Altered expression of genes involved in programmed cell death in primary cultured rat cerebellar granule cells acutely challenged with tetrabromobisphenol A.

In the present study, primary cultures of rat cerebellar granule cells (CGC) and the RT2 Profiler PCR array were used to examine the effect of acutely applied brominated flame retardant tetrabromobisphenol A (TBBPA) on the expression of 84 genes related to the main modes of programmed cell death. CGC, at the 7th day of culture, were exposed to 10 or 25µM TBBPA for 30min. Then, 3, 6, and 24h later, the viability of the cells was examined by the staining with propidium iodide (PI) or using the calcein/ethidium homodimer (CA/ET) live/dead kit, and RNA was extracted for the evaluation of gene expression by RT-PCR. At 3, 6 and 24h after the treatment, the number of viable neurons decreased, according to the PI staining method, to 75%, 58% and 41%, respectively, and with the CA/ET method to 65%, 58% and 28%, respectively. In CGC analyzed 3h after the treatment with 25µM TBBPA or 6h after 10µM TBBPA, the only change in the gene expression was a reduction in the expression of Tnf, which is associated with autophagy and may activate some pro-apoptotic proteins. Six hours after 25µM TBBPA, only 2 genes were over-expressed, a pro-apoptotic Tnfrsf10b and Irgm, which is related to autophagy, and the genes that were suppressed included the anti-apoptotic gene Xiap, the necrosis-related Commd4, pro-apoptotic Abl1, 5 genes involved in autophagy (App, Atg3, Mapk8, Pten, and Snca) and 2 genes that participate in two metabolic pathways: Atp6v1g2 (pro-apoptotic and necrosis) and Tnf (pro-apoptotic, autophagy). Autophagy-related Snca and Tnf remained under-expressed 24h after treatment with 25µM TBBPA, which was accompanied by the over-expression of the pro-apoptotic Casp6, the anti-apoptotic Birc3, 2 genes related to autophagy (Htt and Irgm) and 2 genes (Fas and Tp53) that are involved in both apoptosis (pro-apoptotic) and autophagy. These results show a complex pattern of TBBPA-evoked changes in the expression of the genes involved in the programmed neuronal death, indicating no induction of programmed necrosis, an early suppression of the autophagy and anti-apoptotic genes, followed by a delayed activation of genes associated with apoptosis.

Tetrabromobisphenol A disturbs zinc homeostasis in cultured cerebellar granule cells: A dual role in neurotoxicity.

The brominated flame retardant tetrabromobisphenol A (TBBPA) has recognized neurotoxic properties mediated by intracellular Ca2+ imbalance and oxidative stress. Although these factors are known to trigger the release of Zn2+ from intracellular stores, the effects of TBBPA on Zn2+ homeostasis in neurons and the role of Zn2+in TBBPA neurotoxicity have not yet been studied. Therefore, we investigated zinc transients in primary cultures of rat cerebellar granule cells and assessed their involvement in TBBPA neurotoxicity. The results demonstrate that TBBPA releases Zn2+ from the intracellular stores and increases its intracellular concentration, followed by Zn2+ displacement from the cells. TBBPA-evoked Zn2+ transients are partially mediated by Ca2+ and ROS. Application of TPEN, Zn2+ chelator, potentiates TBBPA- and glutamate-induced 45Ca uptake, enhances TBBPA-induced ROS production and potentiates decreases in the ΔΨm in cells treated with 25 µM TBBPA, revealing the potential neuroprotective capacity of endogenous Zn2+. However, the administration of TPEN does not aggravate TBBPA neurotoxicity, and even slightly decreases neuronal death induced by 25 µM TBBPA. In summary, it was shown for the first time that TBBPA interferes with the cellular Zn2+ homeostasis in neuronal cultures, and we revealed complex roles for endogenous Zn2+ in cytoprotection and TBBPA toxicity in cultured neurons.

The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A.

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges.

Select putative neurodevelopmental toxins modify SNAP-25 expression in primary cultures of rat cerebellar granule cells.

A presynaptic protein SNAP-25 belonging to SNARE complex which is instrumental in intracellular vesicular trafficking and exocytosis, has been implicated in hyperactivity and cognitive abilities in some neuropsychiatric disorders. The unclear etiology of the behavior disrupting neurodevelopmental disabilities in addition to genetic causes most likely involves environmental factors. The aim of this in vitro study was to test if various suspected developmental neurotoxins can alter SNAP-25 mRNA and protein expression in neurons. Real-time PCR and Western blotting analyses were used to assess SNAP-25 mRNA and protein levels in primary cultures of rat cerebellar granule cells (CGCs). The test substances: tetrabromobisphenol-A (TBBPA), thimerosal (TH), silver nanoparticles (NAg), valproic acid (VPA) and thalidomide (THAL), were administered to CGC cultures at subtoxic concentrations for 24h. The results demonstrated that SNAP-25 mRNA levels were increased by 49 and 66% by TBBPA and THAL, respectively, whereas VPA and NAg reduced these levels to 48 and 64% of the control, respectively. The SNAP-25 protein content in CGCs was increased by 79% by TBBPA, 25% by THAL and 21% by NAg; VPA and TH reduced these levels to 73 and 69% of the control, respectively. The variety of changes in SNAP-25 expression on mRNA and protein level suggests the diversity of the mechanism of action of the test substances. This initial study provided no data on concentration-effect relations and on functional changes in CGCs. However it is the first to demonstrate the effect of different compounds that are suspected of causing neurodevelopmental disabilities on SNAP-25 expression. These results suggest that this protein may be a common target for not only inherited but also environmental modifications linked to behavioral deficits in neurodevelopmental disabilities.

Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.

One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-κB (Nf-κB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.

Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study.

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125µΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 µM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture.