Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50>2000 mg/kg): results of an ECVAM validation study.

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92-96%) but relatively low specificity (40-44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.

Exposure-driven risk assessment: applying exposure-based waiving of toxicity tests under REACH.

The REACH Regulation 1907/2006/EC aims to improve knowledge of the potential risks to humans and the environment of the large number of chemicals produced and used in the EU. The testing requirements are likely to trigger numerous toxicological studies, potentially involving millions of experimental animals, despite the professed goal of REACH to reduce vertebrate testing. It may be necessary therefore to shift emphasis away from animal studies towards more pragmatic strategies, reserving animal tests for the substances of greatest concern. One approach is to waive certain tests based on levels of exposure to the substance. This review explores application of 'Exposure-Based Waiving' (EBW) of toxicity studies, with a particular focus on inhalation where possible, considering the potential qualitative and quantitative supporting arguments that might be made, including the use of thresholds of toxicological concern. Incorporating EBW into intelligent testing strategies for substance registration could advance the goals of REACH and the 3Rs (reduction, replacement and refinement of animals in research) by reducing the usage of animals in toxicity tests, whilst maintaining appropriate protection of human health and the environment. However greater regulatory evaluation, acceptance and guidance are required for EBW to achieve its full impact.

A selective, non-peptide caspase-1 inhibitor, VRT-018858, markedly reduces brain damage induced by transient ischemia in the rat.

Numerous preclinical studies have reported neuroprotective effects of new agents in animal studies. None of these agents has yet translated into a successful clinical trial and therefore to a new therapy. There are many possible reasons for this failure, including poor design of clinical trials, mismatch between preclinical and clinical protocols, and insufficient preclinical data. The enzyme caspase-1 has been implicated in neuronal death. Deletion of the caspase-1 gene, or administration of partially selective inhibitors, reduces neuronal injury induced by cerebral ischemia in rodents. We report here, for the first time, that VRT-018858, the non-peptide, active metabolite of the selective caspase-1 inhibitor pro-drug, pralnacasan, markedly reduced ischemic injury in rats. VRT-018858 was neuroprotective when delivered at 1 and 3h (42% and 58% neuroprotection, respectively) but not 6h after injury, and protection was sustained 7 days after the induction of ischemia (66% neuroprotection). These data confirm caspase-1 as an important target for intervention in acute CNS injury, and propose a new class of caspase-1 inhibitors as highly effective neuroprotective agents.

Insulin-like growth factor-1-dependent maintenance of neuronal metabolism through the phosphatidylinositol 3-kinase-Akt pathway is inhibited by C2-ceramide in CAD cells.

Ceramide is a lipid second-messenger generated in response to stimuli associated with neurodegeneration that induces apoptosis, a mechanism underlying neuronal death in Parkinson's disease. We tested the hypothesis that insulin-like growth factor-1 (IGF-1) could mediate a metabolic response in CAD cells, a dopaminergic cell line of mesencephalic origin that differentiate into a neuronal-like phenotype upon serum removal, extend processes resembling neurites, synthesize abundant dopamine and noradrenaline and express the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, and that this process was phosphatidylinositol 3-kinase (PI 3-K)-Akt-dependent and could be inhibited by C(2)-ceramide. The metabolic response was evaluated as real-time changes in extracellular acidification rate (ECAR) using microphysiometry. The IGF-1-induced ECAR response was associated with increased glycolysis, determined by increased NAD(P)H reduction, elevated hexokinase activity and Akt phosphorylation. C(2)-ceramide inhibited all these changes in a dose-dependent manner, and was specific, as it was not induced by the inactive C(2)-ceramide analogue C(2)-dihydroceramide. Inhibition of the upstream kinase, PI 3-K, also inhibited Akt phosphorylation and the metabolic response to IGF-1, similar to C(2)-ceramide. Decreased mitochondrial membrane potential occurred after loss of Akt phosphorylation. These results show that IGF-1 can rapidly modulate neuronal metabolism through PI 3-K-Akt and that early metabolic inhibition induced by C(2)-ceramide involves blockade of the PI 3-K-Akt pathway, and may compromise the first step of glycolysis. This may represent a new early event in the C(2)-ceramide-induced cell death pathway that could coordinate subsequent changes in mitochondria and commitment of neurons to apoptosis.

Integrin-binding RGD peptides induce rapid intracellular calcium increases and MAPK signaling in cortical neurons.

Integrins mediate cell adhesion to the extracellular matrix and initiate intracellular signaling. They play key roles in the central nervous system (CNS), participating in synaptogenesis, synaptic transmission and memory formation, but their precise mechanism of action remains unknown. Here we show that the integrin ligand-mimetic peptide GRGDSP induced NMDA receptor-dependent increases in intracellular calcium levels within seconds of presentation to primary cortical neurons. These were followed by transient activation and nuclear translocation of the ERK1/2 mitogen-activated protein kinase. RGD-induced effects were reduced by the NMDA receptor antagonist MK801, and ERK1/2 signaling was specifically inhibited by ifenprodil and PP2, indicating a functional connection between integrins, Src and NR2B-containing NMDA receptors. GRGDSP peptides were not significantly neuroprotective against excitotoxic insults. These results demonstrate a previously undescribed, extremely rapid effect of RGD peptide binding to integrins on cortical neurons that implies a close, functionally relevant connection between adhesion receptors and synaptic transmission.

Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free radical release.

Interleukin (IL)-1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL-1-induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL-1 on rat cortical cell cultures. Treatment with IL-1beta did not affect the viability of pure cortical neurones. However, IL-1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL-1 was prevented by pre-treatment with the IL-1 receptor antagonist, the broad spectrum caspase inhibitor Boc-Asp-(OMe)-CH(2)F or the antioxidant alpha-tocopherol. The NMDA receptor antagonist dizolcipine (MK-801) attenuated cell death induced by low doses of IL-1beta but the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester had no effect on neuronal cell death induced by IL-1beta. Thus, IL-1 activates the IL-1 type 1 receptor in astrocytes to induce caspase-dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.

The role of inflammation in CNS injury and disease.

For many years, the central nervous system (CNS) was considered to be 'immune privileged', neither susceptible to nor contributing to inflammation. It is now appreciated that the CNS does exhibit features of inflammation, and in response to injury, infection or disease, resident CNS cells generate inflammatory mediators, including proinflammatory cytokines, prostaglandins, free radicals and complement, which in turn induce chemokines and adhesion molecules, recruit immune cells, and activate glial cells. Much of the key evidence demonstrating that inflammation and inflammatory mediators contribute to acute, chronic and psychiatric CNS disorders is summarised in this review. However, inflammatory mediators may have dual roles, with detrimental acute effects but beneficial effects in long-term repair and recovery, leading to complications in their application as novel therapies. These may be avoided in acute diseases in which treatment administration might be relatively short-term. Targeting interleukin (IL)-1 is a promising novel therapy for stroke and traumatic brain injury, the naturally occurring antagonist (IL-1ra) being well tolerated by rheumatoid arthritis patients. Chronic disorders represent a greater therapeutic challenge, a problem highlighted in Alzheimer's disease (AD); significant data suggested that anti-inflammatory agents might reduce the probability of developing AD, or slow its progression, but prospective clinical trials of nonsteroidal anti-inflammatory drugs or cyclooxygenase inhibitors have been disappointing. The complex interplay between inflammatory mediators, ageing, genetic background, and environmental factors may ultimately regulate the outcome of acute CNS injury and progression of chronic neurodegeneration, and be critical for development of effective therapies for CNS diseases.

Metabolic activity: a novel indicator of neuronal survival in the murine dopaminergic cell line CAD.

Apoptosis is implicated in many neurodegenerative diseases, including Parkinson's disease (PD). Neuroprotective strategies targeting apoptosis need to preserve functional integrity of the saved cells to be effective. The aim of the present study was to evaluate a novel approach for analyzing neuronal function that monitors cellular metabolic responses to receptor activation using the microphysiometer. N-Acetyl-sphingosine (C2-ceramide) induced cell death of the neuronal cell line, Cath.a-differentiated (CAD) cells, which resemble catecholaminergic cells of the CNS, and provide a useful in vitro model for the cells affected in PD. C2-ceramide also suppressed the metabolic response of CAD cells to muscarinic receptor activation. Pretreatment with the caspase inhibitor Boc-Asp-(OMe)-fluoromethylketone (BAF) plus neurotrophin-3 (NT-3) reduced C2- ceramide-induced CAD cell death, delaying cell death more effectively than either agent alone; and, most significantly, BAF and NT-3 enabled the cells remaining 24 h after toxin treatment to generate a normal metabolic response to the muscarinic agonist carbachol. On the basis of these results, we suggest that measuring metabolic responses to receptor activation is a useful method for following neuronal viability after toxin treatment and that the combination of caspase inhibitors and neurotrophic factors might be a plausible strategy for improving neuronal survival, with critical preservation of metabolic function.

Activation of integrin alpha5beta1 delays apoptosis of Ntera2 neuronal cells.

Integrins are dynamic membrane proteins that mediate adhesion of cells to the extracellular matrix. Integrins initiate signal transduction, alone and cooperatively with growth factor receptors, and regulate many aspects of cell behavior. We report here that alpha5beta1-mediated adhesion of Ntera2 neuronal cells to fibronectin decreased apoptosis in response to serum withdrawal. Adhesion induced phosphorylation of FAK, and strongly increased the AKT phosphorylation induced by growth factors, demonstrating for the first time in neuronal cells that integrin-mediated adhesion and growth factors cooperate to regulate AKT activity. Integrins exist on cells in different activation states, and cell survival on fibronectin was enhanced by the antibody 12G10, that modulates the conformation of beta1 in favor of its active form. The antibody 12G10 specifically delayed loss of phosphorylation of AKT on serine 473, and GSK-3beta on serine 9, induced by serum withdrawal, suggesting that these kinases are critical sensors of integrin activation on neuronal cells.

Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling.

Cannabinoids (CBs) are neuroprotective in vivo and in vitro, but the mechanisms of their actions are unknown. The aim of this study was to elucidate the signaling pathways that mediate the protective effect of CBs on primary cultured neurons. The neurotoxin S-AMPA induced significant death of rat primary cortical neurons, which was inhibited by the CB agonist HU-210. Antagonists selective for CB(1) or CB(2) receptors (AM 281 or AM 630, respectively) reversed the neuroprotective effect of HU-210 on S-AMPA-induced cell death. HU-210 triggered activation of AKT, but not activation of the ERK1/2, JNK or p38 signaling pathways. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors LY294002 and wortmannin prevented phosphorylation of AKT in response to HU-210, and reversed the neuroprotective effect of HU-210 on S-AMPA-induced excitotoxicity. Thus the PI 3-K/AKT signaling pathway mediates the neuroprotective effect of exogenous cannabinoids such as HU-210 in primary CNS neurons.

CNS injury: the role of the cytokine IL-1.

Injury to the central nervous system (CNS) and the resulting neuronal loss contribute significantly to morbidity and mortality in human and domestic animal populations. Most insults induce inflammation and the expression of cytokines. The specific roles of these proteins in neurological damage and repair are not completely understood. However, members of the interleukin-1 (IL-1) family have clear therapeutic potential: the IL-1 agonists, IL-1 alpha and IL-1 beta, are induced by CNS injury, and central injection of IL-1 increases, whilst peripheral or central administration of the IL-1 antagonist, IL-1ra, reduces the extent of the damage by more than 50%. The mechanism of action of these cytokines is the subject of intense research. In this review, we summarise approaches that are being used to investigate neuronal cell death, and the contribution of inflammation and cytokines, in particular the IL-1 family, to neurodegenerative disorders.

Endogenous interleukin-1 receptor antagonist mediates anti-inflammatory and neuroprotective actions of cannabinoids in neurons and glia.

Interleukin-1 receptor antagonist (IL-1ra) is an important anti-inflammatory cytokine that blocks all known actions of IL-1 and markedly protects against experimentally induced ischemic, excitotoxic, and traumatic brain insults. Cannabinoids (CBs) also exert potent anti-inflammatory and neuroprotective effects, but the mechanisms of their actions are unknown. Here we tested the hypothesis that the actions of CBs are mediated by endogenous IL-1ra. We report for the first time that both CB1 and CB2 receptors modulate release of endogenous IL-1ra from primary cultured glial cells. Activation of CB1 or CB2 receptors increased lipopolysaccharide-induced IL-1ra release, and specific CB1 or CB2 antagonists blocked lipopolysaccharide-induced production of IL-1ra from glial cells. Comparison of neuronal cultures from wild-type mice and mice lacking IL-1ra (knock-out) indicates that endogenous IL-1ra is essential for the neuro-protective effects of CBs against excessive activation of glutamate receptors (excitotoxicity) in response to S-AMPA or NMDA. Similarly, analysis of mixed glial cultures from IL-1ra knock-out mice indicates that endogenous IL-1ra is required for the CB-induced inhibition of nitric oxide production in response to bacterial lipopolysaccharide. These data suggest a novel neuroprotective mechanism of action for CBs in response to inflammatory or excitotoxic insults that is mediated by both CB1 and CB2 receptor-dependent pathways.

Involvement of caspases and calpains in cerebrocortical neuronal cell death is stimulus-dependent.

1. Caspases and calpains are mediators of apoptotic cell death. The objective of this study was to determine the role of caspases and calpains in primary cerebrocortical neuronal (CCN) death in response to a range of stimuli which reportedly induce neuronal apoptosis. 2. Cell death of primary cultures of rat CCN was induced by staurosporine (STS), C2-ceramide (CER), camptothecin (CMT), hydrogen peroxide (H(2)O(2)) or N-methyl-D-aspartate (NMDA). Caspase and calpain activity were assessed by cleavage of alpha-fodrin or fluorogenic substrates. 3. Cell death was analysed by lactate dehydrogenase (LDH) assay in the absence or presence of the pan-caspase inhibitor Boc-Asp-(OMe)-Fluoromethylketone (Baf) and/or the calpain inhibitor calpeptin (CP). Cell death induced by STS, CER or CMT was accompanied by chromatin condensation and activation of multiple caspases, particularly caspase-3-type proteases. Hydrogen peroxide (H(2)O(2)) treatment was accompanied by activation of caspases -1, -6 and -8, but not -3, whereas none of the caspases tested were activated in response to NMDA. 4. With the exception of H(2)O(2), when cell death was accompanied by caspase activation, it was significantly suppressed by Baf. 5. All stimuli also induced calpain activation, but calpeptin only suppressed cell death induced by H(2)O(2). Furthermore, co-treatment with Baf and calpeptin did not alter the cell death relative to either inhibitor alone. 6. These findings suggest the existence of stimulus-dependent routes for the activation of caspases and calpains during death of cortical neurones and imply that although caspases and calpains are activated, their involvement in the execution of cell death varies with the stimulus.