RESUMO
Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.
Assuntos
Células Epiteliais , Rim , Humanos , Transporte de Elétrons/genética , Estudos Prospectivos , Rim/metabolismo , Linhagem Celular , Células Epiteliais/metabolismoRESUMO
Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.
Assuntos
Complexo I de Transporte de Elétrons , Mitocôndrias , Transporte de Elétrons , Células Hep G2 , Hepatócitos , HumanosRESUMO
Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3ß4, and α4ß2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2-3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4ß2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.
Assuntos
Imidazolinas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Nitrocompostos/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Alcenos/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Neonicotinoides/metabolismo , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitrocompostos/metabolismo , Oócitos , Praguicidas/metabolismo , Praguicidas/farmacologia , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Transporte de Elétrons/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Praguicidas/toxicidade , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Proteostase/efeitos dos fármacos , Medição de Risco , TranscriptomaRESUMO
Neonicotinoid pesticides, originally developed to target the insect nervous system, have been reported to interact with human receptors and to activate rodent neurons. Therefore, we evaluated in how far these compounds may trigger signaling in human neurons, and thus, affect the human adult or developing nervous system. We used SH-SY5Y neuroblastoma cells as established model of nicotinic acetylcholine receptor (nAChR) signaling. In parallel, we profiled dopaminergic neurons, generated from LUHMES neuronal precursor cells, as novel system to study nAChR activation in human post-mitotic neurons. Changes of the free intracellular Ca2+ concentration ([Ca2+]i) were used as readout, and key findings were confirmed by patch clamp recordings. Nicotine triggered typical neuronal signaling responses that were blocked by antagonists, such as tubocurarine and mecamylamine. Pharmacological approaches suggested a functional expression of α7 and non-α7 nAChRs on LUHMES cells. In this novel test system, the neonicotinoids acetamiprid, imidacloprid, clothianidin and thiacloprid, but not thiamethoxam and dinotefuran, triggered [Ca2+]i signaling at 10-100 µM. Strong synergy of the active neonicotinoids (at low micromolar concentrations) with the α7 nAChR-positive allosteric modulator PNU-120596 was observed in LUHMES and SH-SY5Y cells, and specific antagonists fully inhibited such signaling. To provide a third line of evidence for neonicotinoid signaling via nAChR, we studied cross-desensitization: pretreatment of LUHMES and SH-SY5Y cells with active neonicotinoids (at 1-10 µM) blunted the signaling response of nicotine. The pesticides (at 3-30 µM) also blunted the response to the non-α7 agonist ABT 594 in LUHMES cells. These data show that human neuronal cells are functionally affected by low micromolar concentrations of several neonicotinoids. An effect of such signals on nervous system development is a toxicological concern.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Neonicotinoides/toxicidade , Praguicidas/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/patologia , Relação Dose-Resposta a Droga , Humanos , Neonicotinoides/administração & dosagem , Neuroblastoma/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.
Assuntos
Agroquímicos/toxicidade , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Desacopladores/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Consumo de Oxigênio/efeitos dos fármacosRESUMO
In the original publication of the article.
RESUMO
Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
Assuntos
Documentação , Processamento Eletrônico de Dados/legislação & jurisprudência , Regulamentação Governamental , Testes de Toxicidade , Toxicologia/legislação & jurisprudência , Animais , Células Cultivadas , Europa (Continente) , Humanos , Formulação de Políticas , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Terminologia como Assunto , Peixe-Zebra/embriologiaRESUMO
Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose â galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.
Assuntos
Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Células-Tronco Neurais/efeitos dos fármacos , Síndromes Neurotóxicas/diagnóstico , Testes de Toxicidade/métodos , Metabolismo dos Carboidratos , Meios de Cultura , Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Galactose/metabolismo , Galactose/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Células-Tronco Neurais/ultraestrutura , Neuritos/efeitos dos fármacos , Desacopladores/toxicidadeRESUMO
Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.
Assuntos
Modelos Biológicos , Mutagênicos/toxicidade , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Teratogênicos/toxicidade , Toxicologia/métodos , Alternativas aos Testes com Animais/tendências , Animais , Automação Laboratorial , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Células Cultivadas , Guias como Assunto , Ensaios de Triagem em Larga Escala/normas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutagênicos/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurotoxinas/metabolismo , Medição de Risco/tendências , Teratogênicos/metabolismo , Testes de Toxicidade Aguda/normas , Toxicocinética , Toxicologia/tendênciasRESUMO
Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.
Assuntos
Rotas de Resultados Adversos , Ecotoxicologia/métodos , Animais , Ecotoxicologia/história , História do Século XXI , Humanos , Camundongos Endogâmicos C57BL , Controle de Qualidade , Medição de Risco/métodos , Biologia de Sistemas , Toxicocinética , Compostos de Vinila/efeitos adversosRESUMO
The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.
Assuntos
Exposição Ambiental/efeitos adversos , Síndromes Neurotóxicas/etiologia , Medição de Risco/métodos , Animais , Humanos , Síndromes Neurotóxicas/fisiopatologiaRESUMO
Acrylamide (ACR) is a known neurotoxicant and developmental neurotoxicant. As a soft electrophile, ACR reacts with thiol groups in cysteine. One hypothesis of ACR induced neurotoxicity and developmental neurotoxicity (DNT) is conjugation with reduced glutathione (GSH) leading to GSH depletion, increased reactive oxygen species (ROS) production and further oxidative stress and cellular damage. In this regard, we have investigated the effect of ACR on neuronal differentiation, glutathione levels and ROS production in the human neuroblastoma SH-SY5Y cell model. After 9 days of differentiation and exposure, ACR significantly impaired area neurites per cell at non-cytotoxic concentrations (0.33 µM and 10 µM). Furthermore, 10 µM ACR dysregulated 9 mRNA markers important for neuronal development, 5 of them being associated with cytoskeleton organization and axonal guidance. At the non-cytotoxic concentrations that significantly attenuate neuronal differentiation, ACR did neither decrease the level of GSH or total glutathione levels, nor increased ROS production. In addition, the expression of 5 mRNA markers for cellular stress was assessed with no significant altered regulation after ACR exposure up to 320 µM. Thus, ACR-induced DNT is not due to GSH depletion and increased ROS production, neither at non-cytotoxic nor cytotoxic concentrations, in the SH-SH5Y model during differentiation.
Assuntos
Acrilamida , Neuroblastoma , Humanos , Espécies Reativas de Oxigênio/metabolismo , Acrilamida/toxicidade , Neuroblastoma/metabolismo , Estresse Oxidativo , Glutationa/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular TumoralRESUMO
Methylmercury (MeHg) is a developmental neurotoxicant, and one potential mechanism of MeHg toxicity is epigenetic dysregulation. In a recent meta-analysis of epigenome-wide association studies (EWAS), associations between prenatal MeHg exposure and DNA methylation at several genomic sites were identified in blood from newborns and children. While EWASs reveal human-relevant associations, experimental studies are required to validate the relationship between exposure and DNA methylation changes, and to assess if such changes have implications for gene expression. Herein, we studied DNA methylation and gene expression of five of the top genes identified in the EWAS meta-analysis, MED31, MRPL19, GGH, GRK1, and LYSMD3, upon MeHg exposure in human SH-SY5Y cells exposed to 8 or 40 nM of MeHg during differentiation, using bisulfite-pyrosequencing and qPCR, respectively. The concentrations were selected to cover the range of MeHg concentrations in cord blood (2-8.5 µg/L) observed in the cohorts included in the EWAS. Exposure to MeHg increased DNA methylation at MED31, a transcriptional regulator essential for fetal development. The results were in concordance with the epidemiological findings where more MED31 methylation was associated with higher concentrations of MeHg. Additionally, we found a non-significant decrease in DNA methylation at GGH, which corresponds to the direction of change observed in the EWAS, and a significant correlation of GGH methylation with its expression. In conclusion, this study corroborates some of the EWAS findings and puts forward candidate genes involved in MeHg's effects on the developing brain, thus highlighting the value of experimental validation of epidemiological association studies.
RESUMO
The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.
Assuntos
Embrião não Mamífero , Peixe-Zebra , Alternativas aos Testes com Animais , Animais , Desenvolvimento Embrionário , Humanos , Mamíferos , Neonicotinoides/toxicidade , Nicotina/toxicidadeRESUMO
There is a worldwide concern on adverse health effects of dietary exposure to acrylamide (AA) due to its presence in commonly consumed foods. AA is formed when carbohydrate rich foods containing asparagine and reducing sugars are prepared at high temperatures and low moisture conditions. Upon oral intake, AA is rapidly absorbed and distributed to all organs. AA is a known human neurotoxicant that can reach the developing foetus via placental transfer and breast milk. Although adverse neurodevelopmental effects have been observed after prenatal AA exposure in rodents, adverse effects of AA on the developing brain has so far not been studied in humans. However, epidemiological studies indicate that gestational exposure to AA impair foetal growth and AA exposure has been associated with reduced head circumference of the neonate. Thus, there is an urgent need for further research to elucidate whether pre- and perinatal AA exposure in humans might impair neurodevelopment and adversely affect neuronal function postnatally. Here, we review the literature with emphasis on the identification of critical knowledge gaps in relation to neurodevelopmental toxicity of AA and its mode of action and we suggest research strategies to close these gaps to better protect the unborn child.
Assuntos
Acrilamida/toxicidade , Exposição Dietética/efeitos adversos , Síndromes Neurotóxicas/embriologia , Acrilamida/farmacocinética , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Manipulação de Alimentos , Humanos , Troca Materno-Fetal , GravidezRESUMO
Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides rotenoids and strobilurins each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts including transcriptomics in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.
Assuntos
Síndromes Neurotóxicas , Praguicidas , Animais , Simulação por Computador , Humanos , Síndromes Neurotóxicas/etiologia , Medição de Risco , IncertezaRESUMO
The objective of the EU-funded integrated project ACuteTox is to develop a strategy in which general cytotoxicity, together with organ-specific toxicity and biokinetic features, are used for the estimation of human acute systemic toxicity. Our role in the project is to characterise the effect of reference chemicals with regard to neurotoxicity. We studied cell membrane potential (CMP), noradrenalin (NA) uptake, acetylcholine esterase (AChE) activity, acetylcholine receptor (AChR) signalling and voltage-operated calcium channel (VOCC) function in human neuroblastoma SH-SY5Y cells after exposure to 23 pharmaceuticals, pesticides or industrial chemicals. Neurotoxic alert chemicals were identified by comparing the obtained data with cytotoxicity data from the neutral red uptake assay in 3T3 mouse fibroblasts. Furthermore, neurotoxic concentrations were correlated with estimated human lethal blood concentrations (LC50). The CMP assay was the most sensitive assay, identifying eight chemicals as neurotoxic alerts and improving the LC50 correlation for nicotine, lindane, atropine and methadone. The NA uptake assay identified five neurotoxic alert chemicals and improved the LC50 correlation for atropine, diazepam, verapamil and methadone. The AChE, AChR and VOCC assays showed limited potential for detection of acute toxicity. The CMP assay was further evaluated by testing 36 additional reference chemicals. Five neurotoxic alert chemicals were generated and orphendrine and amitriptyline showed improved LC50 correlation. Due to the high sensitivity and the simplicity of the test protocol, the CMP assay constitutes a good candidate assay to be included in an in vitro test strategy for prediction of acute systemic toxicity.
Assuntos
Testes de Toxicidade Aguda/métodos , Acetilcolinesterase/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Potenciais da Membrana/efeitos dos fármacos , Neuroblastoma , Norepinefrina/metabolismo , Receptores Colinérgicos/metabolismoRESUMO
Acrylamide (ACR) is a known neurotoxicant which crosses the blood-brain barrier, passes the placenta and has been detected in breast milk. Hence, early-life exposure to ACR could lead to developmental neurotoxicity. The aim of this study was to elucidate if non-cytotoxic concentrations of ACR alter neuronal differentiation by studying gene expression of markers significant for neurodevelopment in the human neuroblastoma SH-SY5Y cell model. Firstly, by using RNASeq we identified two relevant pathways that are activated during 9 days of retinoic acid (RA) induced differentiation i.e. RA receptor (RAR) activation and the cAMP response element-binding protein (CREB) signalling pathways. Next, by qPCR we showed that 1 and 70 µM ACR after 9 days exposure alter the expression of 13 out of 36 genes in the RAR activation pathway and 18 out of 47 in the CREB signalling pathway. Furthermore, the expression of established neuronal markers i.e. BDNF, STXBP2, STX3, TGFB1 and CHAT were down-regulated. Decreased protein expression of BDNF and altered ratio of phosphorylated CREB to total CREB were confirmed by western blot. Our results reveal that micromolar concentrations of ACR sustain proliferation, decrease neurite outgrowth and interfere with signalling pathways involved in neuronal differentiation in the SH-SY5Y cell model.
Assuntos
Acrilamida/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tretinoína/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neuroblastoma/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Toxicological and pharmacological information from human cells and tissues provides knowledge readily applicable to human safety assessment and to the efficacy assessment of pharmaceuticals. The 3R principle in animal studies includes the use of human material in the R of Replacement. The Reduction and Refinement Rs are related to animal use. Knowledge of the 3Rs and successful 3R methods are a prerequisite for the Reduction of animal experiments in the future. More collaboration among researchers using experimental animals and those working in vitro is necessary with mutual respect. The OECD Guidelines for the Testing of Chemicals have included the animal-free part of the 3Rs in guidances for the development and reporting of Adverse Outcome Pathways (AOPs), which is to be part of the Integrated Approaches to Testing and Assessment (IATA). The 3R centres established to help fulfil the Directive 2010/63/EU play an important role to promote the 3Rs and in the development of animal-free toxicology. Research centres in each Nordic country are founded upon solid research activities in cell and organ toxicity, including major EU programmes to promote 3Rs and implementation of good practices and methods broadly in all stakeholders of industry, regulators and academia. In the light of this, the Nordic Symposium on Toxicology and Pharmacology without Animal Experiments addressed more adopted/modified test guidelines or new test guidelines for new end-points, or hazard challenges, new in vitro 3D models, speeding up transfer of knowledge from research to regulation to understand AOP and towards IATA.