Effects of chlorpyrifos and chlorpyrifos-methyl on the outgrowth of axon-like processes, tubulin, and GAP-43 in N2a cells

The aim of this work was to study the neurodegenerative effects of the organophosphate (OP) pesticides chlorpyrifos (CPF) and chlorpyrifos-methyl (CHM) on cultured mouse N2a neuroblastoma cells. CPF or CHM, at a subcytotoxic concentration of 3 μM, were added to the cells either at the time of the induction of cell differentiation (codifferentiation) or 16 h after the induction of differentiation (postdifferentiation). CPF and CHM were similar in inhibiting significantly the outgrowth of axon-like processes from N2a cells after only 4 h exposure under both co- and postdifferentiation exposure conditions. Densitometric scanning of Western blots of extracts of cells treated with CPF or CHM for 4 h revealed significantly decreased cross-reactivity with a monoclonal antibody recognizing the protein GAP-43 under post- but not under codifferentiation exposure conditions. Exposure to CPF or CHM for 4 h under postdifferentiation conditions also resulted in reduced fluorescence of N2a cell body staining with anti-GAP-43. Cross-reactivity of Western blots with a monoclonal antibody recognizing -tubulin was not significantly affected by OP treatment. These data indicate that a disturbance in GAP-43 may be important in the retraction of axons in predifferentiated N2a cells and support the notion that the mechanisms involved in CPF-and CHM-induced inhibition of axonal outgrowth may be different under co- and postdifferentiation exposure conditions

The toxicity of chlorpyrifos towards differentiating mouse N2a neuroblastoma cells

The aim of this work was to study the effects of chlorpyrifos (CPF) on the outgrowth of axons by differentiating mouse N2a neuroblastoma cells. This was achieved by morphological, Western blotting and enzymatic analyses of cells induced to differentiate in the presence and absence of CPF added either at the same time (co-differentiation) or 16 h after (post-differentiation) the induction of cell differentiation. The outgrowth of axon-like processes was impaired following 4 or 8 h exposure to CPF in both co- and post-differentiation experiments. Western blotting analysis revealed reduced levels of neurofilament heavy chain (NF-H) following 8 h of exposure but no significant effect at 4 h under both co- and post-differentiation conditions. By contrast, levels of the heat shock protein HSP-70 were raised at both time points, but only in co-differentiation experiments. Neuropathy target esterase (NTE) activity was lower than controls following 4 or 8 h of exposure under co-differentiation conditions, but not under any post-differentiation conditions. The results suggest that the inhibition of axon production and maintenance by CPF in differentiating N2a cells may involve multiple targets, which are different under co- and post-differentiation conditions

Diazoxon disrupts the expression and distribution of βIII-tubulin and MAP 1B in differentiating N2a cells

This study aimed at assessing the effects of diazoxon (DZO), a major metabolite of the insecticide diazinon (DZ), on key cytoskeletal proteins in differentiating N2a neuroblastoma cells. Initial experiments established that sub-lethal concentrations of 1, 5 and 10 μM DZO produced profound inhibition of neurite outgrowth. Densitometric scanning of probed immunoblots of N2a cell lysates demonstrated that DZO had no effect on total β-tubulin levels. However, probing with a monoclonal antibody that recognised specifically the βIII-tubulin isotype revealed that 10 μM DZO induced a significant reduction in the levels of this particular form. Levels of polyglutamylated tubulin were not altered. Exposure to 10 μM DZO also decreased the expression of microtubule associated protein 1B (MAP 1B). However, DZO had no effect on the expression of MAP tau. DZO also failed to affect the levels neurofilament light (NFL) and neurofilament medium (NFM) chain levels. Indirect immunofluorescence demonstrated that the staining of neurites in treated cells was weaker than in the controls for βIII-tubulin. In conclusion, DZO disrupts the microtubule (MT) network affecting the expression and distribution of two specific MT proteins known to be important in neuritogenesis. DZO may contribute to the developmental neurotoxicity seen following exposure to DZ

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop

ALTEX - Alternatives to Animal ExperimentationCopyright © 2022 the author(s). On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project “Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework” aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.The CIAO project is steered by the Joint Research Centre of the European Commission (EC-JRC), the Humane Society International (HSI), and the Physicians Committee for Responsible Medicine (PCRM). For Jorid Birkelund Sørli, the research is supported by FIKA, Focused Research Effort on Chemicals in the Working Environment from the Danish Government. For Daniel Jacobson, this work was supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC for the US Department of Energy (LOIS:10074) and the National Institutes of Health 3RF1AG053303-01S2