Inhibition of neurite outgrowth in differentiating mouse N2a neuroblastoma cells by phenyl saligenin phosphate: Effects on MAP kinase (ERK 1/2) activation, neurofilament heavy chain phosphorylation and neuropathy target esterase activity

Sub-lethal concentrations of the organophosphate phenyl saligenin phosphate (PSP) inhibited the outgrowth of axon-like processes in differentiating mouse N2a neuroblastoma cells (IC50 2.5 μM). A transient rise in the phosphorylation state of neurofilament heavy chain (NFH) was detected on Western blots of cell extracts treated with 2.5 μM PSP for 4 h compared to untreated controls, as determined by a relative increase in reactivity with monoclonal antibody Ta51 (anti-phosphorylated NFH) compared to N52 (anti-total NFH). However, cross-reactivity of PSP-treated cell extracts was lower than that of untreated controls after 24 h exposure, as indicated by decreased reactivity with both antibodies. Indirect immunofluorescence analysis with these antibodies revealed the appearance of neurofilament aggregates in the cell bodies of treated cells and reduced axonal staining compared to controls. By contrast, there was no significant change in reactivity with anti-a tubulin antibody B512 at either time point. The activation state of the MAP kinase ERK 1/2 increased significantly after PSP treatment compared to controls, particularly at 4 h, as indicated by increased reactivity with monoclonal antibody E-4 (anti-phosphorylated MAP kinase) but not with polyclonal antibody K-23 (anti-total MAP kinase). The observed early changes were concomitant with almost complete inhibition of the activity of neuropathy target esterase (NTE), one of the proposed early molecular targets in organophosphate-induced delayed neuropathy (OPIDN)

Biochemical effects of selective pesticides in mice and rats

The research undertaken in the framework of this Ph.D. thesis aimed mainly at elucidating the biochemical mechanisms involved in the neurotoxic actions of organophosphate and carbamate pesticides. Exposure to these compounds constitutes one of the main causes of poisoning in non-target animal species. The organophosphate chlorpyrifos (CPF) and the carbamate carbaryl (CB) were selected for the study, since these substances represent two of the most extensively used pesticides worldwide. The neurotoxicity of the CB and CPF was assessed in terms of the effects that these compounds exert on both axonal- and synaptic-related events. Accordingly, this research work focused on the effects of CB and CPF on axonal development and neurotransmission. The investigation of the effects of CB and CPF on the process of axon formation involved the use of differentiating mouse neuroblastoma N2a cells in culture. The test compounds were initially assessed in terms of their ability to inhibit the development of axons from N2a cells. CB and CPF were subsequently evaluated in relation to their action on relevant biochemical parameters. Such parameters included: a. the neurofilament heavy chain (neurofilament high molecular weight protein subunit, NF-H) levels, b. the α-tubulin levels, c. the growth associated axonal protein-43 (GAP-43) levels, and d. the activity of the enzyme neuropathy target esterase (NTE). Determination of NF-H, α-tubulin and GAP-43 levels was attained by Western blotting analysis of lysed cell extracts, whereas NTE activity was measured spectrophotometrically. Preliminary experiments, employing the spectrophotometric method of MTT reduction, showed that a final concentration of 3 μΜ for each pesticide has no effect on the viability of N2a cells and this concentration was, therefore, subsequently adopted throughout the course of the study. The results showed that both CB and CPF are capable of reducing significantly the number of the axons extended by differentiating N2a cells after 4 and 8 hours. Western blot analysis indicated that CPF causes a reduction in NF-H levels after 8 hours, whereas CB exerts no effect. α-tubulin levels were shown to be unaffected by either pesticide exposure. On the other hand, both test compounds induce an increase in the expression of GAP-43 after 4 hours. However, GAP-43 levels were found to decrease after 8 hours exposure to CPF. Finally, CPF was shown to inhibit significantly NTE activity after 4 and 8 hours, whereas CB effects NTE inhibition only after 8 hours.Η έρευνα που διενεργήθηκε στα πλαίσια της διδακτορικής αυτής διατριβής είχε ως πρωταρχικό στόχο τη διαλεύκανση των βιοχημικών φαινομένων και μηχανισμών που ενέχονται στην τοξική δράση που ασκούν επιλεγμένα οργανοφωσφορικά και καρβαμιδικά παρασιτοκτόνα στο νευρικό σύστημα των ζώων. Τα παρασιτοκτόνα αυτά συνιστούν μία από τις κυριότερες αιτίες δηλητηριάσεων στα ζώα. Για την πραγματοποίηση της έρευνας επιλέχθηκαν το οργανοφωσφορικό chlorpyrifos (CPF) και το καρβαμιδικό carbaryl (CB), δεδομένου ότι οι ουσίες αυτές αποτελούν δύο από τα συχνότερα χρησιμοποιούμενα παρασιτοκτόνα παγκοσμίως. Στην παρούσα διατριβή μελετήθηκαν οι νευροτοξικές δράσεις του CB και του CPF τόσο στο επίπεδο του νευράξονα όσο και σε αυτό της σύναψης. Στα πλαίσια αυτά, εξετάστηκε η επίδραση των παραπάνω ουσιών σε βιοχημικές παραμέτρους που συνδέονται αφενός με τη διαδικασία της ανάπτυξης των αξόνων και αφετέρου με τη διαδικασία της νευροδιαβίβασης. Η μελέτη της επίδρασης των δύο υπό εξέταση ουσιών στη διαδικασία σχηματισμού των αξόνων έγινε με τη χρησιμοποίηση καλλιεργειών υπό διαφοροποίηση νευροβλαστωματικών κυττάρων Ν2a από μυ. Αρχικά, προσδιορίστηκε ποσοτικά, με τη βοήθεια μικροσκοπίου, η ικανότητα του CB και του CPF να προκαλούν αναστολή της ανάπτυξης των αξόνων από τα κύτταρα Ν2a και ακολούθησε η εξέταση της επίδρασης των δύο παρασιτοκτόνων σε σχετικές βιοχημικές παραμέτρους. Οι παράμετροι αυτές ήταν: α. τα επίπεδα της βαριάς αλύσου των νευρικών νηματίων του κυτταρικού σκελετού (πρωτεΐνη NF-H), β. τα επίπεδα της α-τουμπουλίνης των μικροσωληνίσκων του κυτταρικού σκελετού, γ. τα επίπεδα της πρωτεΐνης ανάπτυξης του νευράξονα-43 (GAP-43) και δ. η δραστικότητα του ενζύμου «εστεράση που αποτελεί στόχο νευροπάθειας» (ΝΤΕ). Ο προσδιορισμός των επιπέδων των πρωτεϊνών NF-H, α-τουμπουλίνης και GAP-43 έγινε με ανοσοχημικές τεχνικές ανάλυσης πρωτεϊνών κατά Western και τη χρήση κατάλληλων μονοκλωνικών αντισωμάτων, ενώ ο προσδιορισμός της δραστικότητας της ΝΤΕ έγινε με φασματοφωτομετρική μέθοδο. Σε προκαταρκτικά πειράματα δείχθηκε, με τη χρήση της φασματοφωτομετρικής μεθόδου της αναγωγής της ουσίας ΜΤΤ, ότι η συγκέντρωση των δύο παρασιτοκτόνων που χρησιμοποιήθηκε κατά τον κύριο πειραματισμό (3 μΜ) δεν επηρεάζει τη βιωσιμότητα των κυττάρων κάτω από τις συνθήκες επώασης που εφαρμόστηκαν

Development of the Adverse Outcome Pathway (AOP): Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities of children

The Adverse Outcome Pathways (AOPs) are designed to provide mechanistic understanding of complex biological systems and pathways of toxicity that result in adverse outcomes (AOs) relevant to regulatory endpoints. AOP concept captures in a structured way the causal relationships resulting from initial chemical interaction with biological target(s) (molecular initiating event) to an AO manifested in individual organisms and/or populations through a sequential series of key events (KEs), which are cellular, anatomical and/or functional changes in biological processes. An AOP provides the mechanistic detail required to support chemical safety assessment, the development of alternative methods and the implementation of an integrated testing strategy. An example of the AOP relevant to developmental neurotoxicity (DNT) is described here following the requirements of information defined by the OECD Users' Handbook Supplement to the Guidance Document for developing and assessing AOPs. In this AOP, the binding of an antagonist to glutamate receptor N-methyl-Daspartate (NMDAR) receptor is defined as MIE. This MIE triggers a cascade of cellular KEs including reduction of intracellular calcium levels, reduction of brain derived neurotrophic factor release, neuronal cell death, decreased glutamate presynaptic release and aberrant dendritic morphology. At organ level, the above mentioned KEs lead to decreased synaptogenesis and decreased neuronal network formation and function causing learning and memory deficit at organism level, which is defined as the AO. There are in vitro, in vivo and epidemiological data that support the described KEs and their causative relationships rendering this AOP relevant to DNT evaluation in the context of regulatory purposes.JRC.F.3-Chemicals Safety and Alternative Method

Toward a Better Testing Paradigm for Developmental Neurotoxicity: OECD Efforts and Regulatory Considerations

Characterization of potential chemical-induced developmental neurotoxicity (DNT) hazard is considered for risk assessment purposes by many regulatory sectors. However, due to test complexity, difficulty in interpreting results and need of substantial resources, the use of the in vivo DNT test guidelines has been limited and animal data on DNT are scarce. To address challenging endpoints such as DNT, the Organisation for Economic Co-Operation and Development (OECD) chemical safety program has been working lately toward the development of integrated approaches for testing and assessment (IATA) that rely on a combination of multiple layers of data (e.g., in vitro, in silico and non-mammalian in vivo models) that are supported by mechanistic knowledge organized according to the adverse outcome pathway (AOP) framework. In 2017, the OECD convened a dedicated OECD expert group to develop a guidance document on the application and interpretation of data derived from a DNT testing battery that relies on key neurodevelopmental processes and is complemented by zebrafish assays. This review will provide a brief overview of the OECD DNT project and summarize various achievements of relevance to the project. The review also presents an opportunity to describe considerations for uptake of the DNT in an in vitro battery in a regulatory context

Developing and applying the adverse outcome pathway concept for understanding and predicting neurotoxicity.

The Adverse Outcome Pathway (AOP) concept has recently been proposed to support a paradigm shift in regulatory toxicology testing and risk assessment. This concept is similar to the Mode of Action (MOA), in that it describes a sequence of measurable key events triggered by a molecular initiating event in which a stressor interacts with a biological target. The resulting cascade of key events includes molecular, cellular, structural and functional changes in biological systems, resulting in a measurable adverse outcome. Thereby, an AOP ideally provides information relevant to chemical structure-activity relationships as a basis for predicting effects of structurally similar compounds. AOPs could potentially also form the basis for qualitative and quantitative predictive modeling of the human adverse outcome resulting from molecular initiating or other key events for which higher-throughput testing methods are available or can be developed. A variety of cellular and molecular processes are known to be critical for normal function of the central (CNS) and peripheral nervous systems (PNS). Because of the biological and functional complexity of the CNS and PNS, it has been challenging to establish causative links and quantitative relationships between key events that comprise the pathways leading from chemical exposure to an adverse outcome in the nervous system. Following introduction of the principles of MOA and AOPs, examples of potential or putative adverse outcome pathways specific for developmental or adult neurotoxicity are summarized and aspects of their assessment considered. Their possible application in developing mechanistically informed Integrated Approaches to Testing and Assessment (IATA) is also discussed

Summary of discussions from 2019 OECD Conference on RNAi Based Pesticides

RNA interference (RNAi) is a biological process in which double-stranded ribonucleic acid (dsRNA) molecules inhibit protein expression. In recent years, the application of dsRNA has been used in the development of agricultural products for pest control. The 2019 Organisation for Economic Cooperation and Development (OECD) Conference on RNAi Based Pesticides ("the Conference") brought together academic, industry, and government experts in various aspects of RNAi to discuss the current state of knowledge and topics to help in developing considerations for risk assessment. The Conference focused on environment, with some discussion of human health. Along with presentations on the use of dsRNA-based products in agriculture, government regulation, risk assessment, and a background on the Draft OECD Working Paper on "Considerations for the Environmental Risk Assessment of the Application of Sprayed or Externally Applied dsRNA-Based Pesticides" ("OECD Working Paper"), the Conference included panel discussions from presenters at the end of each session and a larger discussion session with Conference participants on the environmental fate of dsRNA, non-target organism (NTO) risk assessment, and human health risk assessment. This paper summarizes input from presenters and Conference participants during these discussions. Key considerations from these discussions have already been incorporated into the OECD Working Paper, that once finalized and published, will facilitate regulators in evaluating externally applied dsRNA-based products for potential environmental risks

Investigating cell type specific mechanisms contributing to acute oral toxicity

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA

Investigating cell type specific mechanisms contributing to acute oral toxicity

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.JRC.F.3-Chemicals Safety and Alternative Method

Application of three approaches for quantitative AOP development to renal toxicity

International audienc

Mechanistic Understanding of the Olfactory Neuroepithelium Involvement Leading to Short-Term Anosmia in COVID-19 Using the Adverse Outcome Pathway Framework

Loss of the sense of smell (anosmia) has been included as a COVID-19 symptom by the World Health Organization. The majority of patients recover the sense of smell within a few weeks postinfection (short-term anosmia), while others report persistent anosmia. Several studies have investigated the mechanisms leading to anosmia in COVID-19; however, the evidence is scattered, and the mechanisms remain poorly understood. Based on a comprehensive review of the literature, we aim here to evaluate the current knowledge and uncertainties regarding the mechanisms leading to short-term anosmia following SARS-CoV-2 infection. We applied an adverse outcome pathway (AOP) framework, well established in toxicology, to propose a sequence of measurable key events (KEs) leading to short-term anosmia in COVID-19. Those KEs are (1) SARS-CoV-2 Spike proteins binding to ACE-2 expressed by the sustentacular (SUS) cells in the olfactory epithelium (OE); (2) viral entry into SUS cells; (3) viral replication in the SUS cells; (4) SUS cell death; (5) damage to the olfactory sensory neurons and the olfactory epithelium (OE). This AOP-aligned approach allows for the identification of gaps where more research should be conducted and where therapeutic intervention could act. Finally, this AOP gives a frame to explain several disease features and can be linked to specific factors that lead to interindividual differences in response to SARS-CoV-2 infection