Bromination Pattern of Hydroxylated Metabolites of BDE-47 Affects Their Potency to Release Calcium from Intracellular Stores in PC12 Cells

BACKGROUND: Brominated flame retardants, including the widely used polybrominated diphenyl ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neurotransmitter release by releasing calcium ions (Ca2+) from intracellular stores at much lower concentrations than its environmentally relevant parent congener BDE-47. Recently, several other hydroxylated BDE-47 metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood. OBJECTIVE AND METHODS: To investigate the neurotoxic potential of other environmentally relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47, BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47-6-OH-BDE-47 (and its methoxylated analog 6-MeO-BDE-47), 6 -OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49--on intracellular Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells. RESULTS: In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced Ca2+ release from intracellular stores, although with different lowest observed effect concentrations (LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum (ER; 5-OH-BDE-47 and 6 -OH-BDE-49) or both ER and mitochondria (6-OH-BDE-47, 3-OH-BDE-47, and 4 -OH-BDE-49). When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we observed lower LOECs for 6-OH-BDE-47 and 4 -OH-BDE-49, as well as for BDE-47. CONCLUSIONS: The present findings demonstrate that hydroxylated metabolites of BDE-47 cause disturbance of the [Ca2+]i. Importantly, shielding of the OH group on both sides with bromine atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE metabolites

Evaluation of neurological effects of cerium dioxide nanoparticles doped with different amounts of zirconium following inhalation exposure in mouse models of Alzheimer’s and vascular disease

Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied. We have assessed the neurological effects of subacute inhalation exposures (4 mg/m3 for 3 h/day, 5 days/week for 4 weeks) to cerium dioxide (CeO2) NPs doped with different amounts of zirconium (Zr, 0%, 27% and 78%), to address the influence of particle redox-activity in the 5xFAD transgenic mouse model of AD. Four weeks post-exposure, effects on behaviour were evaluated and brain tissues were analysed for amyloid-β plaque formation and reactive microglia (Iba-1 staining). Behaviour was also evaluated in concurrently exposed non-transgenic C57BL/6J littermates, as well as in Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice as a model of vascular disease. Markers of inflammation and oxidative stress were evaluated in brain cortex. The brains of the NP-exposed 5xFAD mice revealed no accelerated amyloid-β plaque formation. No significant treatment-related behaviour impairments were observed in the healthy C57BL/6J mice. In the 5xFAD and ApoE-/- models, the NP inhalation exposures did not affect the alternation score in the X-maze indicating absence of spatial working memory deficits. However, following inhalation exposure to the 78% Zr-doped CeO2 NPs changes in forced motor performance (string suspension) and exploratory motor activity (X-maze) were observed in ApoE-/- and 5xFAD mice, respectively. Exposure to the 78% doped NPs also caused increased cortical expression of glial fibrillary acidic protein (GFAP) in the C57BL/6J mice. No significant treatment-related changes neuroinflammation and oxidative stress were observed in the 5xFAD and ApoE-/- mice. Our study findings reveal that subacute inhalation exposure to CeO2 NPs does not accelerate the AD-like phenotype of the 5xFAD model. Further investigation is warranted to unravel whether the redox-activity dependent effects on motor activity as observed in the mouse models of AD and vascular disease result from specific neurotoxic effects of these NPs

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

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

Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders

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

Comparison of different in vitro cell models for the assessment of pesticide-induced dopaminergic neurotoxicity

Biomedical and (neuro) toxicity research on (neuro) degenerative diseases still relies strongly on animal models. However, the use of laboratory animals is often undesirable for both ethical and technical reasons. Current in vitro research thus largely relies on tumor derived- or immortalized cell lines. Notably, the suitability of cell lines for studying neurodegeneration is determined by their intrinsic properties. We therefore characterized PC12, SH-SY5Y, MES23.5 and N27 cells with respect to the presence of functional membrane ion channels and receptors as well as for the effects of five known neurotoxic pesticides on cytotoxicity, oxidative stress and parameters of intracellular calcium homeostasis using a combined alamar Blue/CFDA assay, a H2DCFDA assay and single cell fluorescent (Fura-2) calcium imaging, respectively. Although all pesticides demonstrated a certain level of functional neurotoxicity in the different cell lines, our results also demonstrate considerable differences in intrinsic properties and pesticide-induced effects between the cell lines. This clearly indicates that care should be taken when interpreting (neuro)toxicity data as the chosen cell model may greatly influence the outcome

Comparison of different in vitro cell models for the assessment of pesticide-induced dopaminergic neurotoxicity

Biomedical and (neuro) toxicity research on (neuro) degenerative diseases still relies strongly on animal models. However, the use of laboratory animals is often undesirable for both ethical and technical reasons. Current in vitro research thus largely relies on tumor derived- or immortalized cell lines. Notably, the suitability of cell lines for studying neurodegeneration is determined by their intrinsic properties. We therefore characterized PC12, SH-SY5Y, MES23.5 and N27 cells with respect to the presence of functional membrane ion channels and receptors as well as for the effects of five known neurotoxic pesticides on cytotoxicity, oxidative stress and parameters of intracellular calcium homeostasis using a combined alamar Blue/CFDA assay, a H2DCFDA assay and single cell fluorescent (Fura-2) calcium imaging, respectively. Although all pesticides demonstrated a certain level of functional neurotoxicity in the different cell lines, our results also demonstrate considerable differences in intrinsic properties and pesticide-induced effects between the cell lines. This clearly indicates that care should be taken when interpreting (neuro)toxicity data as the chosen cell model may greatly influence the outcome

Organochlorine Insecticides Lindane and Dieldrin and Their Binary Mixture Disturb Calcium Homeostasis in Dopaminergic PC12 Cells

Current hypotheses link long-term environmental exposure of humans to persistent organochlorine (OC) insecticides lindane (HCH) and dieldrin (HEOD) to the development of neurodegenerative disorders, such as Parkinson’s disease. Primary adverse neurological effects of these insecticides are directed at inhibition of GABA_A and glycine receptors, although GABA-independent effects have also been reported. In this paper we describe the effect of dieldrin and a binary mixture of dieldrin and lindane on a critical parameter of neuronal function and survival, i.e., intracellular calcium homeostasis. The intracellular calcium concentration ([Ca²⁺]_i) has been monitored using real-time single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive dye Fura-2. The results demonstrate that nanomolar concentrations of dieldrin time- and concentration-dependently inhibit depolarization-evoked influx of Ca²⁺. Co-exposure of PC12 cells to a mixture of dieldrin and lindane revealed an additive inhibition of the depolarization-evoked increase in [Ca²⁺]_i, whereas the lindane-induced increase in basal [Ca²⁺]_i is inhibited by dieldrin. The combined findings indicate that dieldrin and binary mixtures of organochlorines affect [Ca²⁺]_i already at concentrations below commonly accepted effect concentrations and close to human internal dose levels. Consequently, current findings illustrate the need to take mixtures of OC insecticides into account in human risk assessment

In vitro dopaminergic neurotoxicity of pesticides : a link with neurodegeneration?

Around the globe, chemical compounds are used to treat or repel pests and plagues that pose a threat to food and feed production. From epidemiological studies, it is known that there is a link between exposure to certain chemical classes of these so-called pesticides and the prevalence of neurodegenerative disorders such as Parkinson's disease in humans. However, which particular compound(s) account for this link or what underlying mechanisms are involved is still largely unresolved. The degenerative process in Parkinson's disease is largely limited to the dopaminergic neurons in the basal ganglia. Cellular mechanisms that are implicated in parkinsonian neurodegeneration include mitochondrial dysfunction, oxidative stress, disturbance of intracellular calcium homeostasis and endoplasmic reticulum (ER) stress. A major characteristic that distinguishes the dopaminergic neurons in the basal ganglia from other dopaminergic neurons is a particular reliance on intracellular calcium for spontaneous activity. Considering the energy consuming nature of maintenance of the intracellular calcium homeostasis and its involvement in life and death of a neuron, this may explain the specific vulnerability of this neuronal population. Despite a large variation in primary mechanism of action it has been demonstrated that pesticides from different classes disturb intracellular calcium homeostasis, thus interfering with intracellular calcium signalling. This relates to altered dopaminergic signalling, disturbed protein homeostasis and increased oxidative stress. Therefore, effects of (mixtures of) pesticides on the intracellular calcium homeostasis may play a role in the development of Parkinson's disease in humans. Although human exposure to pesticides via e.g. food often occurs in complex mixtures, (human) risk assessment is largely based on the assessment of single compounds. The discovery of common modes of action across different classes of pesticides therefore underpins the urgency of development of new models and approaches in risk assessment

Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms.

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