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There is an increasing incidence and prevalence of fatty liver disease in the western world, with steatosis as the most prevalent variant. Known causes of steatosis include exposure to food-borne chemicals, and overconsumption of alcohol, carbohydrates and fat, and it is a well-known side effect of certain pharmaceuticals such as tetracycline, amiodarone and tamoxifen (drug-induced hepatic steatosis). Mechanistic knowledge on chemical-induced steatosis has greatly evolved and has been organized into adverse outcome pathways (AOPs) describing the chain of events from first molecular interaction of a substance with a biological system to the adverse outcome, intrahepatic lipid accumulationIn this study, three known steatosis-inducing pesticides (imazalil, clothianidin, and thiacloprid) were tested for their ability to induce hepatic triglyceride accumulation in the zebrafish (Danio rerio) embryo (ZFE) at 5 days post fertilization, both as single compounds and equipotent binary mixtures. The results indicate that the ZFE is very well applicable as a higher tier testing model to confirm effects in downstream key events in AOPs, that is, chemically-induced triglyceride accumulation in the whole organism and production of visible steatosis. Moreover, dose addition could be concluded for binary mixtures of substances with similar and with dissimilar modes of action.
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In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.
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RIVM convened a workshop on the use of New Approach Methodologies (NAMs) for the ad hoc human health risk assessment of food and non-food products. Central to the workshop were two case studies of marketed products with a potential health concern: the botanical Tabernanthe iboga which is used to facilitate mental or spiritual insight or to (illegally) treat drug addiction and is associated with cardiotoxicity, and dermal creams containing female sex hormones, intended for use by perimenopausal women to reduce menopause symptoms without medical supervision. The workshop participants recognized that data from NAM approaches added valuable information for the ad hoc risk assessment of these products, although the available approaches were inadequate to derive health-based guidance values. Recommendations were provided on how to further enhance and implement NAM approaches in regulatory risk assessment, specifying both scientific and technical aspects as well as stakeholder engagement aspects.
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Seguridad de Productos para el Consumidor , Humanos , Medición de RiesgoRESUMEN
BACKGROUND: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA). OBJECTIVE: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation. METHODS: Compounds were selected from a list of chemicals of which exposure is related to a decrease of the AGD in rats and completed with reference compounds. These chemicals were characterised using specific in vitro transcriptional activation (TA) assays for estrogenic and androgenic properties, leading to a final selection of dienestrol as an ER-agonist and flutamide, linuron, and deltamethrin as AR-antagonists. These compounds were then tested in an in vivo model, i.e. in zebrafish (Danio rerio), using sex ratio in the population as an endpoint in order to confirm their feminising effect and MoA. Ultimately, the fish model was used to test a binary mixture of flutamide and dienestrol. RESULTS: Statistical analysis of the binary mixture of flutamide and dienestrol in the fish sexual development tests (FSDT) with zebrafish supported dose addition.
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Disruptores Endocrinos , Perciformes , Plaguicidas , Masculino , Animales , Ratas , Humanos , Pez Cebra , Flutamida , Dienestrol , Feminización , Desarrollo Sexual , Disruptores Endocrinos/toxicidadRESUMEN
The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.
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Rutas de Resultados Adversos , Inteligencia Artificial , Animales , Humanos , Pruebas de Toxicidad , Medición de Riesgo , BélgicaRESUMEN
Parkinson's disease (PD) is a chronic, progressive and disabling neurodegenerative disorder. The prevalence of PD has risen considerably over the past decades. A growing body of evidence suggest that exposure to environmental toxins, including pesticides, solvents and heavy metals (collectively called toxins), is at least in part responsible for this rapid growth. It is worrying that the current screening procedures being applied internationally to test for possible neurotoxicity of specific compounds offer inadequate insights into the risk of developing PD in humans. Improved screening procedures are therefore urgently needed. Our review first substantiates current evidence on the relation between exposure to environmental toxins and the risk of developing PD. We subsequently propose to replace the current standard toxin screening by a well-controlled multi-tier toxin screening involving the following steps: in silico studies (tier 1) followed by in vitro tests (tier 2), aiming to prioritize agents with human relevant routes of exposure. More in depth studies can be undertaken in tier 3, with whole-organism (in)vertebrate models. Tier 4 has a dedicated focus on cell loss in the substantia nigra and on the presumed mechanisms of neurotoxicity in rodent models, which are required to confirm or refute the possible neurotoxicity of any individual compound. This improved screening procedure should not only evaluate new pesticides that seek access to the market, but also critically assess all pesticides that are being used today, acknowledging that none of these has ever been proven to be safe from a perspective of PD. Importantly, the improved screening procedures should not just assess the neurotoxic risk of isolated compounds, but should also specifically look at the cumulative risk conveyed by exposure to commonly used combinations of pesticides (cocktails). The worldwide implementation of such an improved screening procedure, would be an essential step for policy makers and governments to recognize PD-related environmental risk factors.
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Exposure to pesticides is associated with an increased risk of developing Parkinson's disease (PD). Currently, rodent-based risk assessment studies cannot adequately capture neurodegenerative effects of pesticides due to a lack of human-relevant endpoints targeted at neurodegeneration. Thus, there is a need for improvement of the risk assessment guidelines. Specifically, a mechanistic assessment strategy, based on human physiology and (patho)biology is needed, which can be applied in next generation risk assessment. The Adverse Outcome Pathway (AOP) framework is particularly well-suited to provide the mechanistic basis for such a strategy. Here, we conducted a semi-systematic review in Embase and MEDLINE, focused on neurodegeneration and pesticides, to develop an AOP network for parkinsonian motor symptoms. Articles were labelled and included/excluded using the online platform Sysrev. Only primary articles, written in English, focused on effects of pesticides or PD model compounds in models for the brain were included. A total of 66 articles, out of the 1700 screened, was included. PD symptoms are caused by loss of function and ultimately death of dopaminergic neurons in the substantia nigra (SN). Our literature review highlights that a unique feature of these cells that increases their vulnerability is their reliance on continuous low-level influx of calcium. As such, excess intracellular calcium was identified as a central early Key Event (KE). This KE can lead to death of dopaminergic neurons of the SN, and eventually parkinsonian motor symptoms, via four distinct pathways: 1) activation of calpains, 2) endoplasmic reticulum stress, 3) impairment of protein degradation, and 4) oxidative damage. Several receptors have been identified that may serve as molecular initiating events (MIEs) to trigger one or more of these pathways. The proposed AOP network provides the biological basis that can be used to develop a mechanistic testing strategy that captures neurodegenerative effects of pesticides.
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Rutas de Resultados Adversos , Enfermedad de Parkinson , Trastornos Parkinsonianos , Plaguicidas , Humanos , Enfermedad de Parkinson/metabolismo , Calcio/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Neuronas Dopaminérgicas , Plaguicidas/efectos adversos , Sustancia NegraRESUMEN
Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.
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Dopamine is present in a subgroup of neurons that are vital for normal brain functioning. Disruption of the dopaminergic system, e.g., by chemical compounds, contributes to the development of Parkinson's disease and potentially some neurodevelopmental disorders. Current test guidelines for chemical safety assessment do not include specific endpoints for dopamine disruption. Therefore, there is a need for the human-relevant assessment of (developmental) neurotoxicity related to dopamine disruption. The aim of this study was to determine the biological domain related to dopaminergic neurons of a human stem cell-based in vitro test, the human neural progenitor test (hNPT). Neural progenitor cells were differentiated in a neuron-astrocyte co-culture for 70 days, and dopamine-related gene and protein expression was investigated. Expression of genes specific for dopaminergic differentiation and functioning, such as LMX1B, NURR1, TH, SLC6A3, and KCNJ6, were increasing by day 14. From day 42, a network of neurons expressing the catecholamine marker TH and the dopaminergic markers VMAT2 and DAT was present. These results confirm stable gene and protein expression of dopaminergic markers in hNPT. Further characterization and chemical testing are needed to investigate if the model might be relevant in a testing strategy to test the neurotoxicity of the dopaminergic system.
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Neuronas Dopaminérgicas , Células-Madre Neurales , Humanos , Neuronas Dopaminérgicas/metabolismo , Dopamina/metabolismo , Técnicas de Cocultivo , Astrocitos/metabolismo , Diferenciación Celular/fisiología , Células-Madre Neurales/metabolismoRESUMEN
The most direct effects of inhaled harmful constituents are the effects on the airways. However, inhaled compounds can be rapidly absorbed and subsequently result in systemic effects. For example, e-cigarette vapor has been shown to evoke local effects in the lung, although little is known about subsequent effects in secondary target organs such as the brain. Traditionally, such effects are tested using in vivo models. As an alternative, we have combined two in vitro systems, which are Air-Liquid-Interface (ALI) cultured alveolar cells (A549) and rat primary cortical cultures grown on multi-well microelectrode arrays. This allows us to assess the neurological effects of inhaled compounds. We have used exposure to e-cigarette vapor, containing nicotine, menthol, or vanillin to test the model. Our results show that ALI cultured A549 cells respond to the exposure with the production of cytokines (IL8 and GROalpha). Furthermore, nicotine, menthol, and vanillin were found on the basolateral side of the cell culture, which indicates their translocation. Upon transfer of the basolateral medium to the primary cortical culture, exposure-related changes in spontaneous electrical activity were observed correlating with the presence of e-liquid components in the medium. These clear neuromodulatory effects demonstrate the feasibility of combining continuous exposure of ALI cultured cells with subsequent exposure of neuronal cells to assess neurotoxicity. Although further optimization steps are needed, such a combination of methods is important to assess the neurotoxic effects of inhaled compounds realistically. As such, an approach like this could play a role in future mechanism-based risk assessment strategies.
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Cigarrillo Electrónico a Vapor , Sistemas Electrónicos de Liberación de Nicotina , Ratas , Animales , Nicotina/toxicidad , Cigarrillo Electrónico a Vapor/farmacología , Mentol , Células EpitelialesRESUMEN
Exposure to organophosphate (OP) insecticides has been related to several adverse health effects, including neurotoxicity. The primary insecticidal mode of action of OP insecticides relies on (irreversible) binding to acetylcholine esterase (AChE), with -oxon metabolites having a much higher potency for AChE inhibition than the parent compounds. However, OP insecticides can also have non-AChE-mediated effects, including changes in gene expression, neuroendocrine effects, disruption of neurite outgrowth and disturbance of the intracellular calcium (Ca2+) homeostasis. Since Ca2+ is involved in neurotransmission and neuronal development, our research aimed to assess the effects of two widely used OP insecticides, chlorpyrifos (CPF) and diazinon (DZ) and their respective -oxon metabolites, on intracellular Ca2+ homeostasis in human SH-SY5Y cells and rat primary cortical cultures. Furthermore, we assessed the acute and chronic effects of exposure to these compounds on neuronal network maturation and function in rat primary cortical cultures using microelectrode array (MEA) recordings. While inhibition of AChE appears to be the primary mode of action of oxon-metabolites, our data indicate that both parent OP insecticides (CPF and DZ) inhibit depolarization-evoked Ca2+ influx and neuronal activity at concentrations far below their sensitivity for AChE inhibition, indicating that inhibition of voltage-gated calcium channels is a common mode of action of OP insecticides. Notably, parent compounds were more potent than their oxon metabolites, with exposure to diazinon-oxon (DZO) having no effect on both neuronal activity and Ca2+ influx. Human SH-SY5Y cells were more sensitive to OP-induced inhibition of depolarization-evoked Ca2+ influx than rat cortical cells. Acute exposure to OP insecticides had more potent effects on neuronal activity than on Ca2+ influx, suggesting that neuronal activity parameters are especially sensitive to OP exposure. Interestingly, the effects of DZ and chlorpyrifos-oxon (CPO) on neuronal activity lessened after 48 h of exposure, while the potency of CPF did not differ over time. This suggests that neurotoxicity after exposure to different OPs has different effects over time and occurs at levels that are close to human exposure levels. In line with these results, chronic exposure to CPF during 10 days impaired neuronal network development, illustrating the need to investigate possible links between early-life OP exposure and neurodevelopmental disorders in children and highlighting the importance of non-AChE mediated mechanisms of neurotoxicity after OP exposure.
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Cloropirifos , Insecticidas , Síndromes de Neurotoxicidad , Animales , Humanos , Ratas , Acetilcolinesterasa/metabolismo , Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Diazinón/toxicidad , Insecticidas/toxicidad , Insecticidas/metabolismoRESUMEN
In vivo toxicokinetic studies provide evidence for the translocation and accumulation of nanoparticles (NP) in the brain, thereby causing concern for adverse health effects, particularly for effects following chronic exposure. To date, only few studies investigated the effects of NP exposure on neuronal function in vitro, primarily focusing on short-term effects. The aim of this study was therefore to investigate the effects of two common types of NP, titanium dioxide NP (TiO2NP) and silver NP (AgNP), on neuronal function following acute (0.5 h), sub-chronic (24 h and 48 h) and chronic (14 days) exposure in vitro. Effects of NP exposure on intracellular calcium homeostasis, spontaneous neuronal (network) activity and neuronal network morphology were investigated in rat primary cortical cells using respectively, single-cell microscopy calcium imaging, micro-electrode array (MEA) recordings and immunohistochemistry. Our data demonstrate that high doses of AgNP (≥ 30 µg/mL) decrease calcium influx after 24 h exposure, although neuronal activity is not affected following acute and sub-chronic exposure. However, chronic exposure to non-cytotoxic doses of AgNP (1-10 µg/mL) potently decreases spontaneous neuronal (network) activity, without affecting network morphology and viability. Exposure to higher doses (≥ 30 µg/mL) affects network morphology and is also associated with cytotoxicity. In contrast, acute and sub-chronic exposure to TiO2NP is without effects, whereas chronic exposure only modestly reduces neuronal function without affecting morphology. Our combined findings indicate that TiO2NP exposure is of limited hazard for neuronal function whereas AgNP, in particularly during chronic exposure, has profound effects on neuronal (network) function and morphology.
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Nanopartículas del Metal , Nanopartículas , Ratas , Animales , Plata/toxicidad , Nanopartículas del Metal/toxicidad , Calcio , Titanio/toxicidad , Nanopartículas/toxicidadRESUMEN
BACKGROUND: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs). OBJECTIVES: In this study, we explored a workflow to select and experimentally test multiple compounds as a complex mixture with each of the compounds at or below its no observed adverse effect level (NOAEL), in the same zebrafish embryo model. METHODS: Selection of candidate compounds that potentially induce craniofacial malformations was done using in silico methods-structural similarity, molecular docking, and quantitative structure-activity relationships-applied to a database of chemicals relevant for oral exposure in humans via food (EuroMix inventory, n=1,598). A final subselection was made manually to represent different regulatory fields (e.g., food additives, industrial chemicals, plant protection products), different chemical families, and different MOAs. RESULTS: A final selection of eight compounds was examined in the zebrafish embryo model, and craniofacial malformations were observed in embryos exposed to each of the compounds, thus confirming the developmental toxicity as predicted by the in silico methods. When exposed to a mixture of the eight compounds, each at its NOAEL, substantial craniofacial malformations were observed; according to a dose-response analysis, even embryos exposed to a 7-fold dilution of this mixture still exhibited a slight abnormal phenotype. The cumulative effect of the compounds in the mixture was in accordance with dose addition (added doses of the individual compounds after adjustment for relative potencies), despite different MOAs of the compounds involved. DISCUSSION: This case study of a complex mixture inducing craniofacial malformations in zebrafish embryos shows that dose addition can adequately predicted the cumulative effect of a mixture of multiple substances at low doses, irrespective of the (expected) MOA. The applied workflow may be useful as an approach for CRA in general. https://doi.org/10.1289/EHP9888.
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Mezclas Complejas , Pez Cebra , Animales , Alimentos , Humanos , Simulación del Acoplamiento Molecular , Medición de RiesgoRESUMEN
BACKGROUND: There is an increasing concern about the neurotoxicity of engineered nanomaterials (NMs). To investigate the effects of subchronic oral exposures to SiO2 and CeO2 NMs on Alzheimer's disease (AD)-like pathology, 5xFAD transgenic mice and their C57BL/6J littermates were fed ad libitum for 3 or 14 weeks with control food pellets, or pellets dosed with these respective NMs at 0.1% or 1% (w/w). Behaviour effects were evaluated by X-maze, string suspension, balance beam and open field tests. Brains were analysed for plaque load, beta-amyloid peptide levels, markers of oxidative stress and neuroinflammation. RESULTS: No marked behavioural impairments were observed in the mice exposed to SiO2 or CeO2 and neither treatment resulted in accelerated plaque formation, increased oxidative stress or inflammation. In contrast, the 5xFAD mice exposed to 1% CeO2 for 14 weeks showed significantly lower hippocampal Aß plaque load and improved locomotor activity compared to the corresponding controls. CONCLUSIONS: The findings from the present study suggest that long-term oral exposure to SiO2 or CeO2 NMs has no neurotoxic and AD-promoting effects. The reduced plaque burden observed in the mice following dietary CeO2 exposure warrants further investigation to establish the underlying mechanism, given the easy applicability of this administration method.
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Enfermedad de Alzheimer , Nanoestructuras , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Exposición Dietética , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanoestructuras/toxicidad , Placa Amiloide/inducido químicamente , Dióxido de Silicio/toxicidadRESUMEN
Tobacco use is the leading cause of preventable death worldwide and is highly addictive. Nicotine is the main addictive compound in tobacco, but less is known about other components and additives that may contribute to tobacco addiction. The zebrafish embryo (ZFE) has been shown to be a good model to study the toxic effects of chemicals on the neurological system and thus may be a promising model to study behavioral markers of nicotine effects, which may be predictive for addictiveness. We aimed to develop a testing protocol to study nicotine tolerance in ZFE using a locomotion test with light-dark transitions as behavioral trigger. Behavioral experiments were conducted using three exposure paradigms: (1) Acute exposure to determine nicotine's effect and potency. (2) Pre-treatment with nicotine dose range followed by a single dose of nicotine, to determine which pre-treatment dose is sufficient to affect the potency of acute nicotine. (3) Pre-treatment with a single dose combined with acute exposure to a dose range to confirm the hypothesized decreased potency of the acute nicotine exposure. These exposure paradigms showed that (1) acute nicotine exposure decreased ZFE activity in response to dark conditions in a dose-dependent fashion; (2) pre-treatment with increasing concentrations dose-dependently reversed the effect of acute nicotine exposure; and (3) a fixed pre-treatment dose of nicotine induced a decreased potency of the acute nicotine exposure. This effect supported the induction of tolerance to nicotine by the pre-treatment, likely through neuroadaptation. The interpretation of these effects, particularly in view of prediction of dependence and addictiveness, and suitability of the ZFE model to test for such effects of other compounds than nicotine, are discussed.
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Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.
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Síndromes de Neurotoxicidad , Preparaciones Farmacéuticas , Contaminantes Químicos del Agua , Animales , Escala de Evaluación de la Conducta , Embrión no Mamífero , Humanos , Síndromes de Neurotoxicidad/etiología , Pez CebraRESUMEN
In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.
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Encéfalo/efectos de los fármacos , Ingeniería Química/métodos , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Nanoestructuras/química , Nanoestructuras/toxicidad , Animales , Encéfalo/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Plata/química , Plata/metabolismo , Plata/toxicidad , Titanio/química , Titanio/metabolismo , Titanio/toxicidadRESUMEN
In recent years, the development and implementation of animal-free approaches to chemical and pharmaceutical hazard and risk assessment has taken off. Alternative approaches are being developed starting from the perspective of human biology and physiology. Neural tube closure is a vital step that occurs early in human development. Correct closure of the neural tube depends on a complex interplay between proteins along a number of protein concentration gradients. The sensitivity of neural tube closure to chemical disturbance of signalling pathways such as the retinoid pathway, is well known. To map the pathways underlying neural tube closure, literature data on the molecular regulation of neural tube closure were collected. As the process of neural tube closure is highly conserved in vertebrates, the extensive literature available for the mouse was used whilst considering its relevance for humans. Thus, important cell compartments, regulatory pathways, and protein interactions essential for neural tube closure under physiological circumstances were identified and mapped. An understanding of aberrant processes leading to neural tube defects (NTDs) requires detailed maps of neural tube embryology, including the complex genetic signals and responses underlying critical cellular dynamical and biomechanical processes. The retinoid signaling pathway serves as a case study for this ontology because of well-defined crosstalk with the genetic control of neural tube patterning and morphogenesis. It is a known target for mechanistically-diverse chemical structures that disrupt neural tube closure The data presented in this manuscript will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology.
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Modelos Biológicos , Tubo Neural/crecimiento & desarrollo , Animales , Simulación por Computador , Ectodermo , Desarrollo Embrionario , Humanos , Mesodermo , Ratones , Cresta Neural , Placa Neural , Defectos del Tubo Neural , Notocorda , Biología de Sistemas , Tretinoina/metabolismoRESUMEN
Recently, we reported an in vitro toxicogenomics comparison approach to categorize chemical substances according to similarities in their proposed toxicological modes of action. Use of such an approach for regulatory purposes requires, among others, insight into the extent of biological concordance between in vitro and in vivo findings. To that end, we applied the comparison approach to transcriptomics data from the Open TG-GATEs database for 137 substances with diverging modes of action and evaluated the outcomes obtained for rat primary hepatocytes and for rat liver. The results showed that a relatively small number of matches observed in vitro were also observed in vivo, whereas quite a large number of matches between substances were found to be relevant solely in vivo or in vitro. The latter could not be explained by physicochemical properties, leading to insufficient bioavailability or poor water solubility. Nevertheless, pathway analyses indicated that for relevant matches the mechanisms perturbed in vitro are consistent with those perturbed in vivo. These findings support the utility of the comparison approach as tool in mechanism-based risk assessment.