The BMDC model, a performant cell-based test to assess the sensitizing potential and potency of chemicals including pre/pro-haptens.

BACKGROUND: Chemical-induced allergies at workplace represent a significant occupational health issue. These substances must be properly identified as sensitizers. In previous studies, an original model using mouse bone marrow-derived dendritic cells (BMDC) was developed for this purpose. OBJECTIVES: The aim of this study was to evaluate the predictive capacity of the BMDC model with a large panel of sensitizers (including pre- and pro-haptens) and non-sensitizers. METHODS: The readout from the BMDC model is based on expression levels of six phenotypic markers measured by flow cytometry. RESULTS: The results indicate that 29 of the 37 non-sensitizers, and 81 of the 86 sensitizers were correctly classified compared to the Local Lymph Node Assay (LLNA). Statistical analysis revealed the BMDC model to have a sensitivity of 94%, a specificity of 78%, and an accuracy of 89%. The EC2 (Effective Concentration) values calculated with this model allow sensitizers to be categorized into four classes: extreme, strong, moderate and weak. CONCLUSIONS: These excellent predictive performances show that the BMDC model discriminates between sensitizers and non-sensitizers with outstanding precision equal to or better than existing validated alternative models. Moreover, this model allows to predict sensitization potency of chemicals. The BMDC test could therefore be proposed as an additional tool to assess the sensitizing potential and potency of chemicals.

A new cytometry-based method reveals an accumulation of Nrf2 in dendritic cells exposed to two respiratory sensitizers.

The mechanisms underlying chemical respiratory sensitization are incompletely understood. One of the major cell types involved in this pathology are dendritic cells. In this study, the mechanisms of the NRF2-Keap1 pathway were studied using a bone marrow-derived dendritic cell model exposed to two respiratory sensitizers: ammonium hexachloroplatinate (HCP) and ammonium tetrachloroplatinate (ATCP). Expression levels for two Nrf2-regulated genes, hmox1 and srxn1, were analyzed by real time-quantitative polymerase chain reaction. A flow cytometry-based method was also developed to measure intracellular Nrf2 accumulation in dendritic cells following exposure. Exposure to HCP and ATCP increased both hmox1 and srxn1 gene expression, and was associated with accumulation of Nrf2 protein in cells. Overall, these results show that the respiratory sensitizers, in addition to skin sensitizers, can also induced markers associated with NRF2-Keap1 pathway activation in dendritic cells. This study contributes to a better understanding of the adverse outcome of respiratory sensitization.

Activation of T cells by dendritic cells exposed to a reference sensitizer: Towards a promising model to assess the allergenic potential of chemicals.

BACKGROUND: Low molecular weight chemicals constitute one of the major causes of occupational allergies. European legislation on chemicals recommends limiting the use of in vivo models for assessing the sensitizing potential of chemicals, and encourages the development of integrated alternative methods. An in vitro mouse model of bone marrow-derived dendritic cells (BMDCs) that showed good accuracy (75%) and sensitivity (69%) has previously been developed to assess the sensitizing potential of chemicals. OBJECTIVE: To assess the ability of BMDCs to activate T cells (TCs) in vitro. METHODS: BMDCs pre-exposed to the reference sensitizer ammonium hexachloroplatinate (AHCP) were co-cultured with different subpopulations of TCs. TC activation was assessed by surface marker expression, proliferation, and cytokine release. RESULTS: The results showed significant activation of TCs co-cultured with dendritic cells pre-exposed to AHCP as evaluated by CD124 expression, proliferation, and cytokine secretion. Moreover, the response of TCs appeared to be Th2-oriented. Naive TCs were shown to be involved in this response, and the removal of regulatory TCs did not improve the cell response. CONCLUSIONS: The BMDCs used in this previously developed model appear to have the ability to activate TCs, confirming that the BMDC model represents a reliable assay for assessing the sensitizing potential of chemicals.

In vitro detection of chemical allergens: an optimized assay using mouse bone marrow-derived dendritic cells.

BACKGROUND: Identification of the allergenic potency of chemicals is a key step in the safety assessment process. Predictive assays that require no or few animals are needed. OBJECTIVES: To develop an alternative in vitro mouse bone marrow-derived dendritic cell (BMDC) assay to determine the allergenic potential of chemicals. METHODS: BMDCs were exposed to well-known allergens and to non-allergenic chemicals. Surface marker expression and cytokine release of BMDCs were analysed after treatment. RESULTS: Eleven tested chemicals showed a significant stimulation index (SI) of >1.5 (accuracy, 75%; sensitivity, 69%). The four non-allergens all showed a SI of <1.5. Eight contact allergens tested showed a significant SI of >1.5 (accuracy, 92%; sensitivity, 89%), whereas only two respiratory allergens showed a significant SI of >1.5 (accuracy, 60%; sensitivity, 33%). CONCLUSIONS: The results indicate that the BMDC assay could become a reliable test for assessment of the allergenic potential of chemicals. The next step should include the testing of further chemicals, with the aim of integrating this assay into the toolbox of in vitro methods for the evaluation of the allergenic potential of chemicals.

Evaluation of chromium in red blood cells as an indicator of exposure to hexavalent chromium: An in vitro study.

Chromium(VI) compounds are classified as carcinogenic to humans. Whereas chromium measurements in urine and whole blood (i.e., including plasma) are indicative of recent exposure, chromium in red blood cells (RBC) is attributable specifically to Cr(VI) exposure. Before recommending Cr in RBC as a biological indicator of Cr(VI) exposure, in-vitro studies must be undertaken to assess its reliability. The present study examines the relationship between the chromium added to a blood sample and that subsequently found in the RBC. After incubation of total blood with chromium, RBC were isolated, counted and their viability assessed. Direct analysis of chromium in RBC was conducted using Atomic Absorption Spectrometry. Hexavalent, but not trivalent Cr, was seen to accumulate in the RBC and we found a strong correlation between the Cr(VI) concentration added to a blood sample and the amount of Cr in RBC. This relationship appears to be independent of the chemical properties of the human blood samples (e.g., different blood donors or different reducing capacities). Even though in-vivo studies are still needed to integrate our understanding of Cr(VI) toxicokinetics, our findings reinforce the idea that a single determination of the chromium concentration in RBC would enable biomonitoring of critical cases of Cr(VI) exposure.

H2S induced hypometabolism in mice is missing in sedated sheep.

On the basis of studies performed in mice that showed H(2)S inhalation decreasing dramatically the metabolic rate, H(2)S was proposed as a means of protecting vital organs from traumatic or ischemic episodes in humans. Hypoxia has in fact also long been shown to induce hypometabolism. However, this effect is observed solely in small-sized animals with high VO2 kg(-1), and not in large mammals. Thus, extrapolating the hypometabolic effect of H(2)S to large mammals is questionable and could be potentially dangerous. We measured metabolism in conscious mice (24 g) exposed to H(2)S (60 ppm) at an ambient temperature of 23-24 degrees C. H(2)S caused a rapid and large (50%) drop in gas exchange rate, which occurred independently of the change in body temperature. The metabolic response occurred within less than 3 min. In contrast, sheep, sedated with ketamine and weighing 74 kg did not exhibit any decrease in metabolic rate during a similar challenge at an ambient temperature of 22 degrees C. While a part of H(2)S induced hypometabolism in the mice is related to the reduction in activity, we speculate that the difference between sheep and mice may rely on the nature and the characteristics of the relationship between basal metabolic rate and body weight thus on the different mechanisms controlling resting metabolic rate according to body mass. Therefore, the proposed use of H(2)S administration as a way of protecting vital organs should be reconsidered in view of the lack of hypometabolic effect in a large sedated mammal and of H(2)S established toxicity.

Soluble oligomers of amyloid-beta peptide induce neuronal apoptosis by activating a cPLA2-dependent sphingomyelinase-ceramide pathway.

Recent data have revealed that soluble oligomeric amyloid-beta peptide (Abeta) may be the proximate effectors of neuronal injuries and death in Alzheimer's disease (AD) by unknown mechanisms. Consistently, we recently demonstrated the critical role of a redox-sensitive cytosolic calcium-dependent phospholipase A2 (cPLA2)-arachidonic acid (AA) pathway in Abeta oligomer-induced cell death. According to the involvement of oxidative stress and polyunsaturated fatty acids like AA in the regulation of sphingomyelinase (SMase) activity, the present study underlines the role of SMases in soluble Abeta-induced apoptosis. Soluble Abeta oligomers induced the activation of both neutral and acidic SMases, as demonstrated by the direct measurement of their enzymatic activities, by the inhibitory effects of both specific neutral and acidic SMase inhibitors, and by gene knockdown using antisense oligonucleotides. Furthermore, soluble Abeta-mediated activation of SMases and subsequent cell death were found to be inhibited by antioxidant molecules and a cPLA2-specific inhibitor or antisense oligonucleotide. We also demonstrate that sphingosine-1-phosphate is a potent neuroprotective factor against soluble Abeta oligomer-induced cell death and apoptosis by inhibiting soluble Abeta-induced activation of acidic sphingomyelinase. These results suggest that Abeta oligomers induce neuronal death by activating neutral and acidic SMases in a redox-sensitive cPLA2-AA pathway.

Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers.

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.

Microtubule-associated protein MAP1A, MAP1B, and MAP2 proteolysis during soluble amyloid beta-peptide-induced neuronal apoptosis. Synergistic involvement of calpain and caspase-3.

A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid beta-peptide (Abeta) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble Abeta remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437-3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble Abeta-mediated neuronal apoptosis. The data presented here show that soluble Abeta oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Abeta-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble Abeta. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble Abeta-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.

Cytosolic phospholipase A2 mediates neuronal apoptosis induced by soluble oligomers of the amyloid-beta peptide.

Recent data have revealed that soluble oligomeric forms of amyloid peptide (Abeta) may be the proximate effectors of the neuronal injury and death occurring in Alzheimer's disease (AD). However, the molecular mechanisms associated with the neuronal cell death induced by the nonfibrillar Abeta remain to be elucidated. In this study, we investigated the role of the cytosolic Ca2+-dependent phospholipase A2 (cPLA2), and its associated metabolic pathway, i.e., the arachidonic acid (AA) cascade, in the apoptotic cell death induced by soluble oligomers of Abeta. The treatment of rat cortical neurons with low concentrations of soluble Abeta(1-40) or Abeta(1-42) peptide resulted in an early calcium-dependent release of AA associated with a transient relocalization of cPLA2. Both cPLA2 antisense oligonucleotides and a selective inhibitor of cPLA2 activity abolished the release of AA from neurons and also protected cells against apoptosis induced by Abeta. Furthermore, inhibitors of the PKC, p38, and MEK/ERK pathways that are involved in cPLA2 phosphorylation and activation reduced Abeta-induced cell death. Finally, we demonstrate that inhibitors of cyclooxygenase-2 reduced the Abeta-induced cell death by 55%. Our studies suggest a novel neuronal response of soluble oligomers of Abeta, which occurs through a cPLA2 signaling cascade and an AA-dependent death pathway. This may prove to be crucial in AD processes and could provide important targets for drug development.

Oligodendrocytes are susceptible to apoptotic cell death induced by prion protein-derived peptides.

Neurodegenerative prion diseases, characterized by a progressive dementia, are associated with the accumulation of abnormal forms of the prion (PrPc) protein, potentially due to an aberrant regulation of PrPc biogenesis and/or topology. One of these forms, termed ctmPrP, displays a transmembrane conformation and might trigger neuronal cell death in Gerstmann-Straüssler-Scheinker (GSS) syndrome and other prion-associated diseases in humans. Although the primary target cells involved in the progression of prion diseases remain unidentified, it was recently suggested that modifications of the oligodendroglial cells occur early in prion diseases. In the present study, we demonstrate that a putative transmembrane domain of the human PrPc, i.e., amino acids 118-135, induces oligodendrocyte (OLG) death in vitro in a time- and dose-dependent manner. The process leading to OLG death and induced by the PrP[118-135] peptide was characterized by DNA fragmentation, cytoskeletal disruption, and caspase activation. Protection against the PrP[118-135] peptide-induced OLG apoptosis by several antioxidant molecules, such as probucol, propylgallate, and promethazine, suggests that oxidative injuries contribute to the PrP[118-135] cytotoxicity to OLGs. These results suggest a potential pathophysiological role of the ctmPrP- and/or PrP fragment-mediated OLG cytotoxicity in spongiform encephalopathies.

Membrane cholesterol interferes with neuronal apoptosis induced by soluble oligomers but not fibrils of amyloid-beta peptide.

Neuronal cell death in Alzheimer's disease (AD) is partly induced by the interaction of the amyloid-beta peptide (Abeta) with the plasma membrane of target cells. Accordingly, recent studies have suggested that cholesterol, an important component of membranes that controls their physical properties and functions, plays a critical role in neurodegenerative diseases. We report here that the enrichment of the neuronal plasma membrane with cholesterol protects cortical neurons from apoptosis induced by soluble oligomers of the Abeta(1-40) peptide. Conversely, cholesterol depletion using cyclodextrin renders cells more vulnerable to the cytotoxic effects of the Abeta-soluble oligomers. Increasing the cholesterol content of small unilamellar liposomes also decreases Abeta-dependent liposome fusion. We clearly demonstrate that cholesterol protection is specific to the soluble conformation of Abeta, because we observed no protective effects on cortical neurons treated by amyloid fibrils of the Abeta(1-40) peptide. This may provide a new opportunity for the development of an effective AD therapy as well as elucidate the impact of the cholesterol level during AD development.

Humanin rescues cortical neurons from prion-peptide-induced apoptosis.

We recently demonstrated that a soluble oligomeric prion peptide, the putative 118-135 transmembrane domain of prion protein (PrP), exhibited membrane fusogenic properties and induced apoptotic cell death both in vitro and in vivo. A recently discovered rescue factor humanin (HN) was shown to protect neuronal cells from various insults involved in human neurodegenerative diseases. We thus addressed the question of whether HN might modulate the apoptosis induced by the soluble PrP(118-135) fragment. We found that the incubation of rat cortical neurons with 10 microM HN prevented soluble PrP(118-135) fragment-induced cell death concomitantly with inhibition of apoptotic events. An HN variant, termed HNG, exhibited a 500-fold increase in the protective activity in cortical neurons, whereas the HNA variant displayed no protective effect. The effects of HN and HNG peptides did not require a preincubation with the PrP(118-135) fragment, strongly suggesting that these peptides rescue cells independently of a direct interaction with the prion peptide. By contrast, and in agreement with a previous study, HN had no effect on the fibrillar PrP(106-126) peptide-induced cell death. This protective effect for neurons from PrP(118-135)-induced cell death strongly suggests that PrP(118-135) and PrP(106-126) peptides may trigger different pathways leading to neuronal apoptosis.

In vivo and in vitro neurotoxicity of the human prion protein (PrP) fragment P118-135 independently of PrP expression.

We recently demonstrated that the 118-135 putative transmembrane domain of prion protein (PrP) exhibited membrane fusogenic properties and induced apoptotic neuronal cell death of rat cortical neurons, independently of its aggregation state. The aim of the present study was to analyze the in vivo neurotoxicity of the prion fragment P118-135 and to evaluate the potential role of the physiological isoform of PrP in the P118-135-induced cell death. Here, we demonstrate that the nonfibrillar P118-135 is cytotoxic to retinal neurons in vivo as monitored by intravitreal inoculation and recording of the electrical activity of retina and tissue examination. Moreover, knock-out PrP gene mice exhibit similar sensitivity to the nonfibrillar P118-135-induced cell death and electrical perturbations, strongly suggesting that cell death occurs independently of PrP expression. Interestingly, a variant nonfusogenic P118-135 peptide (termed P118-135theta) had no effects on in vivo neuronal viability, suggesting that the P118-135-induced cell death is mediated by its membrane destabilizing properties. These data have further been confirmed in vitro. We show that the fusogenic peptide P118-135 induces death of cultured neurons from both wild-type and knock-out PrP gene mice via an apoptotic-mediated pathway, involving early caspase activation and DNA fragmentation. Altogether these results emphasize the neurotoxicity of the fusogenic nonfibrillar PrP transmembrane domain and indicate that fibril formation and PrP expression are not obligatory requirements for neuronal cell death. The use of synthetic prion peptides could provide insights into the understanding of neuronal loss mechanisms that take place during the development of the various types of spongiform encephalopathies.

Apoptotic neuronal cell death induced by the non-fibrillar amyloid-beta peptide proceeds through an early reactive oxygen species-dependent cytoskeleton perturbation.

In the present study, we have determined the nature and the kinetics of the cellular events triggered by the exposure of cells to non-fibrillar amyloid-beta peptide (A beta). When cortical neurons were treated with low concentrations of soluble A beta (1-40), an early reactive oxygen species (ROS)-dependent cytoskeleton disruption precedes caspase activation. Indeed, caspase activation and neuronal cell death were prevented by the microtubule-stabilizing drug taxol. A perturbation of the microtubule network was noticeable after being exposed to A beta for 1 h, as revealed by electron microscopy and immunocytochemistry. Microtubule disruption and neuronal cell death induced by A beta were inhibited in the presence of antioxidant molecules, such as probucol. These data highlight the critical role of ROS production in A beta-mediated cytoskeleton disruption and neuronal cell death. Finally, using FRAP (fluorescence recovery after photo bleaching) analysis, we observed a time-dependent biphasic modification of plasma membrane fluidity, as early as microtubule disorganization. Interestingly, molecules that inhibited neurotubule perturbation and cell death did not affect the membrane destabilizing properties of A beta, suggesting that the lipid phase of the plasma membrane might represent the earliest target for A beta. Altogether our results convey the idea that upon interaction with the plasma membrane, the non-fibrillar A beta induces a rapid ROS-dependent disorganization of the cytoskeleton, which results in apoptosis.