Determinants of the essential one-carbon metabolism metabolites, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine and folate, in cerebrospinal fluid.

BACKGROUND: Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population. METHODS: Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used. RESULTS: CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites. CONCLUSIONS: Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.

Use of aggregating cell cultures for toxicological studies.

Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies.

Strategies and tools for preventing neurotoxicity: to test, to predict and how to do it

A change in paradigm is needed in the prevention of toxic effects on the nervous system, moving from its present reliance solely on data from animal testing to a prediction model mostly based on in vitro toxicity testing and in silico modeling. According to the report published by the National Research Council (NRC) of the US National Academies of Science, high-throughput in vitro tests will provide evidence for alterations in"toxicity pathways" as the best possible method of large scale toxicity prediction. The challenges to implement this proposal are enormous, and provide much room for debate. While many efforts address the technical aspects of implementing the vision, many questions around it need also to be addressed. Is the overall strategy the only one to be pursued? How can we move from current to future paradigms? Will we ever be able to reliably model for chronic and developmental neurotoxicity in vitro? This paper summarizes four presentations from a symposium held at the International Neurotoxicology Conference held in Xi"an, China, in June 2011. A. Li reviewed the current guidelines for neurotoxicity and developmental neurotoxicity testing, and discussed the major challenges existing to realize the NCR vision for toxicity testing. J. Llorens reviewed the biology of mammalian toxic avoidance in view of present knowledge on the physiology and molecular biology of the chemical senses, taste and smell. This background information supports the hypothesis that relating in vivo toxicity to chemical epitope descriptors that mimic the chemical encoding performed by the olfactory system may provide a way to the long term future of complete in silico toxicity prediction. S. Ceccatelli reviewed the implementation of rodent and human neural stem cells (NSCs) as models for in vitro toxicity testing that measures parameters such as cell proliferation, differentiation and migration. These appear to be sensitive endpoints that can identify substances with developmental neurotoxic potential. C. Sun ol reviewed the use of primary neuronal cultures in testing for neurotoxicity of environmental pollutants, including the study of the effects of persistent exposures and/or in differentiating cells, which allow recording of effects that can be extrapolated to human developmental neurotoxicity.

Toxic neurofilamentous axonopathies accumulation of neurofilaments and axonal degeneration

A number of neurotoxic chemicals induce accumulation of neurofilaments in axonal swellings that appear at varying distances from the cell body. This pathology is associated with axonal degeneration of different degrees. The clinical manifestation is most commonly that of a mixed motor-sensory peripheral axonopathy with a disto-proximal pattern of progression, as in cases of chronic exposure to n-hexane and carbon disulphide. It has been demonstrated that protein adduct formation is a primary molecular mechanism of toxicity in these axonopathies, but how this mechanism leads to neurofilament accumulation and axonal degeneration remains unclear. Furthermore, little is known regarding the mechanisms of neurofilamentous axonopathy caused by 3,3′-iminodipropionitrile, an experimental toxin that induces proximal axon swelling that is strikingly similar to that found in early amyotrophic lateral sclerosis. Here, we review the available data and main hypotheses regarding the toxic axonopathies and compare them with the current knowledge of the biological basis of neurofilament transport. We also review recent studies addressing the question of how these axonopathies may cause axonal degeneration. Understanding the mechanisms underlying the toxic axonopathies may provide insight into the relationship between neurofilament behaviour and axonal degeneration, hopefully enabling the identification of new targets for therapeutic intervention. Because neurofilament abnormalities are a common feature of many neurodegenerative diseases, advances in this area may have a wider impact beyond toxicological significance

Cerebral Hypoperfusion in Posterior Reversible Encephalopathy Syndrome is Different from Transient Ischemic Attack on CT Perfusion.

BACKGROUND: PRES is a reversible neurotoxic state presenting with headache, altered mental status, visual loss, and seizures. Delayed diagnosis can be avoided if radiological patterns could distinguish PRES from cerebral ischemia. METHODS: Clinical and radiological data were collected on all hospitalized patients who had (1) discharge diagnosis of PRES and (2) acute CTP/CTA. Data were compared with 10 TIA patients with proven cytotoxic edema on MRI. RESULTS: Of the four PRES patients found, three were correlated with acute blood pressure and one with chemotherapy. At the radiological level, quantitative analyses of the CTP parameters showed that 2 out of 4 patients had bilaterally reduced CBF-values (23.2-47.1 ml/100g/min) in occipital regions, as seen in the pathological regions of TIA patients (27.3 ± 13.5 ml/100g/min). When compared with TIA patients, the pathological ROI's demonstrated decreased CBV-values (3.4-5.6 ml/100g). Vasogenic edema on MRI FLAIR imaging was seen in only one PRES patient, and cytotoxic edema on DWI-imaging was never found. CT angiography showed in one PRES patient a vasospasm-like unilateral posterior cerebral artery. CONCLUSIONS: If confirmed by other groups, CTP and CTA imaging in patients with acute visual loss and confusion may help to distinguish PRES from bi-occipital ischemia. These radiological parameters may identify PRES patients at risk for additional tissue infarction.

Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse

Allylnitrile, cis-crotononitrile, and 3,3 -iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5±0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.

Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures.

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

The influence of genetic and environmental factors among MDMA users in cognitive performance

This study is aimed to clarify the association between MDMA cumulative use and cognitive dysfunction, and the potential role of candidate genetic polymorphisms in explaining individual differences in the cognitive effects of MDMA. Gene polymorphisms related to reduced serotonin function, poor competency of executive control and memory consolidation systems, and high enzymatic activity linked to bioactivation of MDMA to neurotoxic metabolites may contribute to explain variations in the cognitive impact of MDMA across regular users of this drug. Sixty ecstasy polydrug users, 110 cannabis users and 93 non-drug users were assessed using cognitive measures of Verbal Memory (California Verbal Learning Test, CVLT), Visual Memory (Rey-Osterrieth Complex Figure Test, ROCFT), Semantic Fluency, and Perceptual Attention (Symbol Digit Modalities Test, SDMT). Participants were also genotyped for polymorphisms within the 5HTT, 5HTR2A, COMT, CYP2D6, BDNF, and GRIN2B genes using polymerase chain reaction and TaqMan polymerase assays. Lifetime cumulative MDMA use was significantly associated with poorer performance on visuospatial memory and perceptual attention. Heavy MDMA users (>100 tablets lifetime use) interacted with candidate gene polymorphisms in explaining individual differences in cognitive performance between MDMA users and controls. MDMA users carrying COMT val/val and SERT s/s had poorer performance than paired controls on visuospatial attention and memory, and MDMA users with CYP2D6 ultra-rapid metabolizers performed worse than controls on semantic fluency. Both MDMA lifetime use and gene-related individual differences influence cognitive dysfunction in ecstasy users.

Métodos de preparação e atividade biológica do ácido quinolínico e derivados

In 1981 2,3-pyridine dicarboxylic acid (quinolinic acid) was discovery to be a selective agonist for the N-methyl -D-aspartic acid (NMDA) receptor. As a consequence it possesses neurotoxic activity resulting from overstimulation of the receptor. Quinolinic acid is implicated as an etiological factor in a range of neurodegenerative disease including AIDS related dementia, Huntington´s disease and Lyme disease. In the design of novel therapies to treat these diseases, some molecules have been identified as an important target. In this paper we described different methods to prepare quinolinic acid and derivatives.

Defesa química: histórico, classificação dos agentes de guerra e ação dos neurotóxicos

Chemical agents are substances used for their toxic effects on humans, animals and plants. The main objective of chemical defense is to develop systems that reduce these effects while minimizing impact on the operational capacity of military troops. In this work, a report on the development of chemical warfare agents since the First World War and their classification is presented. Special attention is given to neurotoxic agents, the most lethal group of chemical agents known to date.

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits ³H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of ³H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM) and up to 10 min of treatment, crotoxin decreased ³H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on ³H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits ³H-choline uptake with high affinity (EC25 = 10 ± 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

Interactions of laminin with the amyloid ß peptide: Implications for Alzheimer's disease

Extensive neuronal cell loss is observed in Alzheimer's disease. Laminin immunoreactivity colocalizes with senile plaques, the characteristic extracellular histopathological lesions of Alzheimer brain, which consist of the amyloid ß (Aß) peptide polymerized into amyloid fibrils. These lesions have neurotoxic effects and have been proposed to be a main cause of neurodegeneration. In order to understand the pathological significance of the interaction between laminin and amyloid, we investigated the effect of laminin on amyloid structure and toxicity. We found that laminin interacts with the Aß1-40 peptide, blocking fibril formation and even inducing depolymerization of preformed fibrils. Protofilaments known to be intermediate species of Aß fibril formation were also detected as intermediate species of laminin-induced Aß fibril depolymerization. Moreover, laminin-amyloid interactions inhibited the toxic effects on rat primary hippocampal neurons. As a whole, our results indicate a putative anti-amyloidogenic role of laminin which may be of biological and therapeutic interest for controlling amyloidosis, such as those observed in cerebral angiopathy and Alzheimer's disease.