Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.

Risks for human health related to the presence of grayanotoxins in certain honey.

The European Commission asked EFSA for a scientific opinion on the risks for human health of the presence of grayanotoxins (GTXs) in 'certain honey' from Ericaceae plants. The risk assessment included all structurally related grayananes occurring with GTXs in 'certain' honey. Oral exposure is associated with acute intoxication in humans. Acute symptoms affect the muscles, nervous and cardiovascular systems. These may lead to complete atrioventricular block, convulsions, mental confusion, agitation, syncope and respiratory depression. For acute effects, the CONTAM Panel derived a reference point (RP) of 15.3 µg/kg body weight for the sum of GTX I and III based on a BMDL10 for reduced heart rate in rats. A similar relative potency was considered for GTX I. Without chronic toxicity studies, an RP for long-term effects could not be derived. There is evidence for genotoxicity in mice exposed to GTX III or honey containing GTX I and III, showing increased levels of chromosomal damage. The mechanism of genotoxicity is unknown. Without representative occurrence data for the sum of GTX I and III and consumption data from Ericaceae honey, acute dietary exposure was estimated based on selected concentrations for GTX I and III reflecting concentrations measured in 'certain' honeys. Applying a margin of exposure (MOE) approach, the estimated MOEs raised health concerns for acute toxicity. The Panel calculated the highest concentrations for GTX I and III below which no acute effects would be expected following 'certain honey' consumption. The Panel is 75% or more certain that the calculated highest concentration of 0.05 mg for the sum of GTX I and III per kg honey is protective for all age groups regarding acute intoxications. This value does not consider other grayananes in 'certain honey' and does not cover the identified genotoxicity.

Short- and Long-Term Effects of Suboptimal Selenium Intake and Developmental Lead Exposure on Behavior and Hippocampal Glutamate Receptors in a Rat Model.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

A Clarion Call for Neuroinflammatory Assays in Brain Stimulation for Chronic Pain.

Chronic pain is characterized by an impaired functional state (pain, mood, sleep, cognition, and metabolism) affecting different brain networks relevant for pain perception and neural pain processing [...].

Immune and Nervous Systems Interaction in Endocrine Disruptors Toxicity: The Case of Atrazine.

Endocrine disruptors (ED) are natural and anthropogenic chemicals that can interfere with hormonal systems at different levels. As such, ED-induced alterations in hormone functions have been implicated in many diseases and pathological conditions, including adverse developmental, reproductive, neurological, cardiovascular, and immunological effects in mammals. The fact that ED may compete with several endogenous hormones for multiple receptors and pathways is not always fully considered. This results in a complex response that depends on the cellular context in terms of receptors and interacting proteins and, thus, may differ between tissues and circumstances. Microglia, neurons, and other immune cells are potential targets and still underappreciated actors in endocrine disruption. Due to the large scale of this topic, this review is not intended to provide a comprehensive review nor a systematic review of chemicals identified as endocrine disruptors. It focuses on the immune-neuro-endocrine network in ED toxicity and research gaps, using atrazine as an example to highlight this complexity and the interrelationship between the immune, endocrine, and nervous systems, and ED.

Neural In Vitro Models for Studying Substances Acting on the Central Nervous System.

Animal models have been greatly contributing to our understanding of physiology, mechanisms of diseases, and toxicity. Yet, their limitations due to, e.g., interspecies variation are reflected in the high number of drug attrition rates, especially in central nervous system (CNS) diseases. Therefore, human-based neural in vitro models for studying safety and efficacy of substances acting on the CNS are needed. Human iPSC-derived cells offer such a platform with the unique advantage of reproducing the "human context" in vitro by preserving the genetic and molecular phenotype of their donors. Guiding the differentiation of hiPSC into cells of the nervous system and combining them in a 2D or 3D format allows to obtain complex models suitable for investigating neurotoxicity or brain-related diseases with patient-derived cells. This chapter will give an overview over stem cell-based human 2D neuronal and mixed neuronal/astrocyte models, in vitro cultures of microglia, as well as CNS disease models and considers new developments in the field, more specifically the use of brain organoids and 3D bioprinted in vitro models for safety and efficacy evaluation.

Sex-Dependent Effects of Developmental Lead Exposure in Wistar Rats: Evidence from Behavioral and Molecular Correlates.

Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains.

Sex differences in steroid levels and steroidogenesis in the nervous system: Physiopathological role.

The nervous system, in addition to be a target for steroid hormones, is the source of a variety of neuroactive steroids, which are synthesized and metabolized by neurons and glial cells. Recent evidence indicates that the expression of neurosteroidogenic proteins and enzymes and the levels of neuroactive steroids are different in the nervous system of males and females. We here summarized the state of the art of neuroactive steroids, particularly taking in consideration sex differences occurring in the synthesis and levels of these molecules. In addition, we discuss the consequences of sex differences in neurosteroidogenesis for the function of the nervous system under healthy and pathological conditions and the implications of neuroactive steroids and neurosteroidogenesis for the development of sex-specific therapeutic interventions.

Corrigendum to Recommendation on test readiness criteria for new approach methods in toxicology: exemplified for developmental neurotoxicity.

In this manuscript, which appeared in ALTEX 35 , 306-352 ( doi:10.14573/altex.1712081 ), the Acknowledgements should read: This work was supported by the Doerenkamp-Zbinden Foundation, EFSA, the BMBF, JPI-NutriCog-Selenius, and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

Evaluation of the health risks related to the presence of cyanogenic glycosides in foods other than raw apricot kernels.

In 2016, the EFSA Panel on Contaminants in the Food Chain (CONTAM) published a scientific opinion on the acute health risks related to the presence of cyanogenic glycosides (CNGs) in raw apricot kernels in which an acute reference dose (ARfD) of 20 µg/kg body weight (bw) was established for cyanide (CN). In the present opinion, the CONTAM Panel concluded that this ARfD is applicable for acute effects of CN regardless the dietary source. To account for differences in cyanide bioavailability after ingestion of certain food items, specific factors were used. Estimated mean acute dietary exposures to cyanide from foods containing CNGs did not exceed the ARfD in any age group. At the 95th percentile, the ARfD was exceeded up to about 2.5-fold in some surveys for children and adolescent age groups. The main contributors to exposures were biscuits, juice or nectar and pastries and cakes that could potentially contain CNGs. Taking into account the conservatism in the exposure assessment and in derivation of the ARfD, it is unlikely that this estimated exceedance would result in adverse effects. The limited data from animal and human studies do not allow the derivation of a chronic health-based guidance value (HBGV) for cyanide, and thus, chronic risks could not be assessed.

Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity.

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia.

In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).

Risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods.

Tetrodotoxin (TTX) and its analogues are produced by marine bacteria and have been detected in marine bivalves and gastropods from European waters. The European Commission asked EFSA for a scientific opinion on the risks to public health related to the presence of TTX and TTX analogues in marine bivalves and gastropods. The Panel on Contaminants in the Food Chain reviewed the available literature but did not find support for the minimum lethal dose for humans of 2 mg, mentioned in various reviews. Some human case reports describe serious effects at a dose of 0.2 mg, corresponding to 4 µg/kg body weight (bw). However, the uncertainties on the actual exposure in the studies preclude their use for derivation of an acute reference dose (ARfD). Instead, a group ARfD of 0.25 µg/kg bw, applying to TTX and its analogues, was derived based on a TTX dose of 25 µg/kg bw at which no apathy was observed in an acute oral study with mice, applying a standard uncertainty factor of 100. Estimated relative potencies for analogues are lower than that of TTX but are associated with a high degree of uncertainty. Based on the occurrence data submitted to EFSA and reported consumption days only, average and P95 exposures of 0.00-0.09 and 0.00-0.03 µg/kg bw, respectively, were calculated. Using a large portion size of 400 g bivalves and P95 occurrence levels of TTX, with exception of oysters, the exposure was below the group ARfD in all consumer groups. A concentration below 44 µg TTX equivalents/kg shellfish meat, based on a large portion size of 400 g, was considered not to result in adverse effects in humans. Liquid chromatography with tandem mass spectroscopy (LC-MS/MS) methods are the most suitable for identification and quantification of TTX and its analogues, with LOQs between 1 and 25 µg/kg.

Dihydrotestosterone as a Protective Agent in Chronic Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis is a chronic inflammatory disease affecting the central nervous system. As reported by clinical observations, variation in hormonal levels might alter disease susceptibility and progression. Specifically, decreased levels of testosterone in males are reported to be permissive for disease onset. Accordingly, testosterone seems to exert protective effects in experimental autoimmune encephalomyelitis (EAE). In this context, it is important to highlight that testosterone is further metabolized into 17ß-estradiol or dihydrotestosterone (DHT). In this study, we aimed to explore the protective effects of DHT treatment in EAE Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction showed that DHT exerts a beneficial effect on clinical scores, coupled with decreased gliosis (i.e. glial fibrillary acidic protein and major histocompatibility complex of class II staining) and inflammation (i.e. translocator protein 18 kDa, interleukin-1ß, Toll-like receptor 4 and nuclear factor-κB expression) in the spinal cord. Moreover, parameters linked to oxidative stress and tissue damage, like thiobarbituric acid-reactive substance levels and Bcl-2-associated X protein expression, and to mitochondrial activity (i.e. content of mitochondrial DNA and proteins), were improved after DHT administration. This neuroactive steroid may be further metabolized into 3α- or 3ß-diol. However, assessment of the levels of these metabolites after DHT treatment seems to suggest that the protective effects observed here are due to DHT itself. Altogether, the present results indicate that DHT was effective in reducing the severity of chronic EAE and, consequently, may represent an interesting perspective for multiple sclerosis treatment.

Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability.

Increasing evidence underlines that prototypical inflammatory cytokines (IL-1ß, TNF-α and IL-6) either synthesized in the central (CNS) or peripheral nervous system (PNS) by resident cells, or imported by immune blood cells, are involved in several pathophysiological functions, including an unexpected impact on synaptic transmission and neuronal excitability. This review describes these unconventional neuromodulatory properties of cytokines, that are distinct from their classical action as effector molecules of the immune system. In addition to the role of cytokines in brain physiology, we report evidence that dysregulation of their biosynthesis and cellular release, or alterations in receptor-mediated intracellular pathways in target cells, leads to neuronal cell dysfunction and modifications in neuronal network excitability. As a consequence, targeting of these cytokines, and related signalling molecules, is considered a novel option for the development of therapies in various CNS or PNS disorders associated with an inflammatory component. This article is part of a Special Issue entitled 'Neuroimmunology and Synaptic Function'.

Perspectives on neuroinflammation and excitotoxicity: a neurotoxic conspiracy?

Emerging evidences underline the ability of several environmental contaminants to induce an inflammatory response within the central nervous system, named neuroinflammation. This can occur as a consequence of a direct action of the neurotoxicant to the CNS and/or as a response secondary to the activation of the peripheral inflammatory response. In both cases, neuroinflammation is driven by the release of several soluble factors among which pro-inflammatory cytokines. IL-1ß and TNF-α have been extensively studied for their effects within the CNS and emerged for their role in the modulation of the neuronal response, which allow the immune response to integrate with specific neuronal functions, as neurotransmission and synaptic plasticity. In particular, it has been evidenced a potential detrimental link between these cytokines and the glutamatergic system that seems to be part of increased brain excitability and excitotoxicity occurring in different pathological conditions. Aim of this mini-review will be to present experimental evidence on the way IL-1ß and TNF-α impact neurons, focusing on the glutamatergic signalling, to provide a perspective on novel pathways possibly involved in environmental contaminants neurotoxicity.

Corticosteroids modulate the expression of the PKC-anchoring protein RACK-1 and cytokine release in THP-1 cells.

We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.

Diabetic neuropathic pain: a role for testosterone metabolites.

Diabetic neuropathy is associated with neuropathic pain in about 50% of diabetic subjects. Clinical management of neuropathic pain is complex and so far unsatisfactory. In this study, we analyzed the effects of the testosterone metabolites, dihydrotestosterone (DHT), and 3α-diol, on nociceptive and allodynia thresholds and on molecular and functional parameters related to pain modulation in the dorsal horns of the spinal cord and in the dorsal root ganglia of rats rendered diabetic by streptozotocin injection. Furthermore, the levels of DHT and 3α-diol were analyzed in the spinal cord. Diabetes resulted in a significant decrease in DHT levels in the spinal cord that was reverted by DHT or 3α-diol treatments. In addition, 3α-diol treatment resulted in a significant increase in 3α-diol in the spinal cord compared with control values. Both steroids showed analgesic properties on diabetic neuropathic pain, affecting different pain parameters and possibly by different mechanisms of action. Indeed, DHT counteracted the effect of diabetes on the mechanical nociceptive threshold, pre- and post-synaptic components, glutamate release, astrocyte immunoreactivity, and expression of interleukin-1ß (IL1ß), while 3α-diol was effective on tactile allodynia threshold, glutamate release, astrocyte immunoreactivity and the expression of substance P, toll-like receptor 4, tumor necrosis factor-α, transforming growth factor ß-1, IL1ß, and translocator protein. These results indicate that testosterone metabolites are potential agents for the treatment of diabetic neuropathic pain.

Levels and actions of progesterone and its metabolites in the nervous system during physiological and pathological conditions.

Progesterone is synthesized and actively metabolized in the central and peripheral nervous system, into neuroactive steroid metabolites, such as dihydroprogesterone, allopregnanolone and isopregnanolone. Progesterone and/or its metabolites exert a variety of effects acting as physiological regulators of neuronal and glial development and plasticity, controlling reproduction, neuroendocrine events, mood and affection. In addition, these neuroactive steroids maintain neural homeostasis and exert neuroprotective actions. In agreement, metabolic pathways of progesterone are affected by modifications in the level of gonadal hormones and by pathology or injury with a regional specificity and in a sex-dimorphic way. Therefore, observations here summarized may provide a background to design sex-specific therapies based on progesterone metabolites. On this point of view, considering that one of the major limits of a therapy based on neuroactive steroids could be modifications in their plasma levels and their consequent peripheral effects, pharmacological treatments aimed to increase their levels in the nervous system could provide an interesting therapeutic option.