Glutamate - A multifaceted molecule: Endogenous neurotransmitter, controversial food additive, design compound for anti-cancer drugs. A critical appraisal.

One of the most widely used flavour enhancers in the food industry is monosodium glutamate (MSG). MSG consumption has been on an upward trend, worrying in terms of potential toxic effects. This review is focused on the long-term toxicity of MSG and the experimental evidence that supports it. The article's primary purpose was to survey recently published data regarding the consumption of MSG within safe limits. The administered doses in animal models are very varied and have given rise to controversy. Also, the paper comprises pathways to lower MSG toxicity and highlight other underexploited biological effects, as anti-cancer potential. The administration of MSG, combined with various compounds, has been shown benefit against toxic effects. Several recent studies have identified a possible mechanism that recommends MSG and some derivatives as potential anti-cancer agents. New anti-cancer compounds based on the glutamic acid structure must be studied and further exploited. International regulations require harmonization of safe doses of MSG based on current scientific studies. Replacing MSG with other umami flavour enhancers may be a safer alternative for human health in the future. The biological consequences of MSG consumption or therapeutical administration have not been fully deciphered yet.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Oral administration of potassium bromate induces neurobehavioral changes, alters cerebral neurotransmitters level and impairs brain tissue of swiss mice.

BACKGROUND: Potassium bromate (KBrO3) is widely used as a food additive and is a major water disinfection by-product. The present study reports the side effects of KBrO3 administration on the brain functions and behaviour of albino mice. METHODS: Animals were divided into three groups: control, low dose KBrO3 (100 mg/kg/day) and high dose KBrO3 (200 mg/kg/day) groups. RESULTS: Administration of KBrO3 led to a significant change in the body weight in the animals of the high dose group in the first, second and the last weeks while water consumption was not significantly changed. Neurobehavioral changes and a reduced Neurotransmitters levels were observed in both KBrO3 groups of mice. Also, the brain level of reduced glutathione (GSH) in KBrO3 receiving animals was decreased. Histological studies favoured these biochemical results showing extensive damage in the histological sections of brain of KBrO3-treated animals. CONCLUSIONS: These results show that KBrO3 has serious damaging effects on the central nervous system and therefore, its use should be avoided.

PACAP in the BNST produces anorexia and weight loss in male and female rats.

Recent gene association studies have implicated pituitary adenylate cyclase-activating peptide (PACAP) systems in several psychiatric disorders associated with stressor exposure, and we have argued that many of the behavioral consequences of repeated stressor exposure may depend on the expression of PACAP in the bed nucleus of the stria terminalis (BNST). One behavioral consequence of the activation of stress systems can be anorexia and subsequent weight loss, and both the activation of central PACAP systems as well as neuronal activity in the BNST have also been associated with anorexic states in rodents. Hence, we investigated the regulation of food and water intake and weight loss following BNST PACAP infusion. BNST PACAP38 dose-dependently decreased body weight, as well as food and water intake in the first 24 h following infusion. Because different BNST subregions differentially regulate stress responding, we further examined the effects of PACAP38 in either the anterior or posterior BNST. Anterior BNST PACAP38 infusion did not alter weight gain, whereas posterior PACAP38 infusion resulted in weight loss. PACAP38 infused into the lateral ventricles did not alter weight, suggesting that the effects of BNST-infused PACAP were not mediated by leakage into the ventricular system. These data suggest that PACAP receptor activation in posterior BNST subregions can produce anorexia and weight loss, and corroborate growing data implicating central PACAP activation in mediating the consequences of stressor exposure.

N-stearoyltyrosine protects against glutamate-induced oxidative toxicity by an apoptosis-inducing factor (AIF)-mediated caspase-independent cell death pathway.

N-stearoyltyrosine (NsTyr), a synthesized anandamide (AEA) analogue, could exert potent neuroprotective effects on cerebral ischemia models both in vivo and in vitro via intervening in multiple injuries. Glutamate, a major excitatory neurotransmitter, plays a critical role during stroke/cerebral ischemia. In this study, we explored the protective effects of NsTyr on glutamate neurotoxicity in PC12 cells and investigated its underlying mechanisms. NsTyr treatment attenuated glutamate-induced oxidative toxicity in a dose-dependent manner and the best performance was observed at 10 µΜ. NsTyr treatment suppressed glutamate-induced upregulation of lipoxygenase 12/15 (LOX 12/15) activity and reactive oxygen species (ROS) elevation, attenuated the increase of BH3-interacting domain death agonist (Bid) in the mitochondria, prevented the loss of mitochondria membrane potential and consequently inhibited apoptosis-inducing factor (AIF) translocation into the nucleus. The results demonstrated that NsTyr could protect cells against AIF-mediated caspase-independent cell death induced by glutamate, which may be due to the blockage of Bid-mediated mitochondrial damage via reducing LOX 12/15 activity and ROS accumulation.

DJ-1 deficiency triggers microglia sensitivity to dopamine toward a pro-inflammatory phenotype that is attenuated by rasagiline.

DJ-1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ-1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ-1 deficiency may increase microglial neurotoxicity. We found that down-regulation of DJ-1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro-inflammatory cytokines such as IL-1ß and IL-6. Furthermore, we discovered that DJ-1-deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro-inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ-1-deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro-inflammation in neurodegenerative diseases. Further studies of DJ-1-mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention

Neuroprotective activity of 2-amino-1,3,4-thiadiazole derivative 4BrABT--an in vitro study.

4BrABT (2-(4-Bromophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole) is a compound known for its interesting in vitro anticancer profile. 4BrABT inhibited proliferation and motility of several cancer cell lines in concentrations which were not toxic to normal cells. A major problem associated with cancer chemotherapy, but also caused by environmental factors such as pesticides, is neurotoxicity. Therefore, the aim of the presented study was an in vitro evaluation of the neuroprotective activity of this compound. 4BrABT activity (1-100 µM) was tested in cultures of mouse neurons, rat astrocytes and rat oligodendrocytes. A possible protective action of the compound in different neurodegenerative models, as serum deprivation (SD), excitotoxicity (presence of 500 µM glutamate in culture medium), as well as cisplatin toxicity (astroglia--50 µM and oligodendroglia--100 µM) was investigated. Cell viability in the tested cultures was assessed with the use of LDH and MTT methods. Moreover, 4BrABT ability to prevent the cisplatin-induced apoptosis in astrocyte and oligodendrocyte cultures was analysed after Hoechst 33342 fluorostaining. The obtained results indicate that 4BrABT was not toxic to neurons, astrocytes and oligodendrocytes. Moreover, a decrease in the neuronal LDH level was observed, which may suggest the ability of 4BrABT to act as a trophic agent. Furthermore, the protective action of the studied compound was shown in neuronal cultures exposed to neurotoxic conditions (presence of glutamate and trophic stress) and in cisplatin-treated astrocytes and oligodendrocytes. The expression of anticancer and neuroprotective activity raises hopes for the potential use of 4BrABT as a safe anticancer drug, or neuroprotective agent in chemotherapy-associated neurotoxicity.

Extracellular hydrogen peroxide contributes to oxidative glutamate toxicity.

Oxidative glutamate toxicity is characterized by the inhibition of cystine uptake, the depletion of intracellular glutathione, and increased levels of intracellular reactive oxygen species, factors that lead to neuronal injury. We found that the presence of extracellular catalase protected cultured neuronal cells, such as HT22, SH-SY5Y and PC12 cells, from glutamate-induced cytotoxicity. Extracellular hydrogen peroxide (H2O2) accumulated in a time- and concentration-dependent manner in HT22 cells during prolonged exposure to glutamate. To investigate the involvement of NADPH oxidase in glutamate-induced H2O2 generation, we used small interference RNA (siRNA). Knockdown of Nox2 and Nox4 expression reduced H2O2 accumulation and increased cell survival. siRNA specific for Nox4 reduced the production of H2O2 by ~74% compared with control siRNA. Furthermore, H2O2 accumulation was also suppressed by U0126, a MEK/ERK inhibitor, in a concentration-dependent manner. These results suggest that glutamate triggers the Nox-dependent generation of extracellular H2O2 via ERK1/2 activation, which contributes to oxidative glutamate toxicity.

Modification with organometallic compounds improves crossing of the blood-brain barrier of [Leu5]-enkephalin derivatives in an in vitro model system.

Enkephalin peptides are thought to be suitable vectors for the passage of the blood-brain barrier (BBB). Modifications that do not alter the amino acid sequence are often used to improve the permeation through living membrane systems. As a new type of modification we introduce organometallic compounds, in particular ferrocene carboxylic acid. Derivatives of [Leu5]enkephalin were synthesised and labelled with organometallic compounds by using solid-phase synthesis techniques. All new metal-peptide bioconjugates were comprehensively characterised by HPLC, NMR spectroscopy and mass spectrometry and found to be at least 95% pure. For the first time, permeation coefficients in a BBB model for organometal-peptide derivatives were determined in this work. The uptake and localisation of fluorescein-labelled enkephalins was monitored by fluorescence microscopy on three cancer cell lines. Octanol/H2O partition coefficients of the compounds were measured by HPLC. The introduction of the organometallic moiety enhances the uptake into cells and the permeation coefficient of [Leu5]-enkephalin. This could be due to an increase in lipophilicity caused by the organometallic label. The metal-peptide conjugates were found to be nontoxic up to mM concentrations. The low cytotoxicity encourages further experiments that could take advantage of the selectivity of enkephalin derivatives for opioid receptors.

Is chemical neurotransmission altered specifically during methylmercury-induced cerebellar dysfunction?

Methylmercury (MeHg) is an important environmental neurotoxicant that is present in seafood and affects the developing and mature nervous system. The neurotoxicity induced by MeHg is a concern, particularly for fish-eating populations and pregnant or nursing women. During MeHg-induced neurotoxicity, degeneration of the granule cell layer in the cerebellum occurs, which leads to deficits in motor function. I suggest that the action of MeHg on specific neurotransmitter receptors contributes to the selective vulnerability of granule cells. MeHg appears to stimulate M(3) muscarinic acetylcholine receptors and to inhibit GABA(A) receptor subtypes preferentially on cerebellar granule cells. This could lead to the loss of tonic inhibition of granule cells as a result of antagonism of GABA(A) receptors, and a M(3)-receptor-mediated increase in the intracellular concentration of Ca(2+) and block of a K(+)-dependent leak current. The net result would be increased spontaneous release of glutamate, which, coupled with a MeHg-induced impairment of glutamate uptake by astrocytes, could cause Ca(2+)-mediated cytotoxicity.

Early pathophysiological features in canine renal papillary necrosis induced by nefiracetam.

To ascertain the early pathophysiological features in canine renal papillary necrosis (RPN) caused by the neurotransmission enhancer nefiracetam, male beagle dogs were orally administered nefiracetam at 300 mg/kg/day for 4 to 7 weeks in comparison with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), at 50 mg/kg/day for 5 weeks. During the dosing period, the animals were periodically subjected to laboratory tests, light-microscopic, immunohistochemical, and electron-microscopic examinations and/or cyclooxygenase (COX)-2 mRNA analysis. In laboratory tests, a decrease in urinary osmotic pressure and increases in urine volume and urinary lactate dehydrogenase (LDH) level were early biomarkers for detecting RPN. Light-microscopically, nefiracetam revealed epithelial swelling and degeneration in the papillary ducts in week 7, while ibuprofen displayed degeneration and necrosis in the papillary interstitium in week 5. In immunohistochemical staining with COX-2 antibody, nefiracetam elicited a positive reaction within interstitial cells around the affected epithelial cells in the papillary ducts (upper papilla) in week 7, and ibuprofen positively reacted within interstitial cells adjacent to the degenerative and/or necrotic lesions in week 5. Ultrastructurally, nefiracetam exhibited reductions of intracellular interdigitation and infoldings of epithelial cells in the papillary ducts, whereas ibuprofen showed no changes in the identical portions. Thus, the early morphological change in the papilla brought about by nefiracetam was quite different from that elicited by ibuprofen. By the renal papillary COX-2 mRNA expression analysis, nefiracetam exceedingly decreased its expression in week 4, but markedly increased it in week 7, suggesting an induction of COX-2 mRNA by renal papillary lesions. These results demonstrate that the epithelial cell in the papillary ducts is the primary target site for the onset of RPN evoked by nefiracetam.

Heterologous desensitization of beta-adrenoceptor signal transduction in vivo.

The hemodynamic actions of pituitary adenylate cyclase-activating polypeptide (PACAP-27) rapidly diminish upon repeated i.v. injection in rats treated with the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). We now report that the beta-adrenoceptor agonist isoproterenol (0.5 microg/kg, i.v.) produces pronounced hypotensive and vasodilator effects in anesthetized rats pretreated with L-NAME (100 micromol/kg, i.v.). However, the hypotensive and vasodilator actions of isoproterenol were markedly diminished in L-NAME-treated rats in which tachyphylaxis to PACAP was induced immediately prior to the injection of the beta-adrenoceptor agonist. This suggests that a reduction in tissue concentrations of nitric oxide-containing factors allows tachyphylaxis to PACAP-27-mediated vasodilation to occur in vivo and that this process leads to the heterologous desensitization of beta-adrenoceptor-mediated responses.

Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain?

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate and/or cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mg/g). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate-induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinate/g protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Protective effects of a vitamin B12 analog, methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons.

The effects of methylcobalamin, a vitamin B12 analog, on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Cell viability was markedly reduced by a brief exposure to glutamate followed by incubation with glutamate-free medium for 1 h. Glutamate cytotoxicity was prevented when the cultures were maintained in methylcobalamin-containing medium. Glutamate cytotoxicity was also prevented by chronic exposure to S-adenosylmethionine, which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and S-adenosylmethionine also inhibited the cytotoxicity induced by N-methyl-D-aspartate or sodium nitroprusside that releases nitric oxide. In cultures maintained in a standard medium, glutamate cytotoxicity was not affected by adding methylcobalamin to the glutamate-containing medium. In contrast, acute exposure to MK-801, a NMDA receptor antagonist, prevented glutamate cytotoxicity. These results indicate that chronic exposure to methylcobalamin protects cortical neurons against NMDA receptor-mediated glutamate cytotoxicity.