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.

Substantiate a read-across hypothesis by using transcriptome data-A case study on volatile diketones.

This case study explores the applicability of transcriptome data to characterize a common mechanism of action within groups of short-chain aliphatic α-, ß-, and γ-diketones. Human reference in vivo data indicate that the α-diketone diacetyl induces bronchiolitis obliterans in workers involved in the preparation of microwave popcorn. The other three α-diketones induced inflammatory responses in preclinical in vivo animal studies, whereas beta and gamma diketones in addition caused neuronal effects. We investigated early transcriptional responses in primary human bronchiolar (PBEC) cell cultures after 24 h and 72 h of air-liquid exposure. Differentially expressed genes (DEGs) were assessed based on transcriptome data generated with the EUToxRisk gene panel of Temp-O-Seq®. For each individual substance, genes were identified displaying a consistent differential expression across dose and exposure duration. The log fold change values of the DEG profiles indicate that α- and ß-diketones are more active compared to γ-diketones. α-diketones in particular showed a highly concordant expression pattern, which may serve as a first indication of the shared mode of action. In order to gain a better mechanistic understanding, the resultant DEGs were submitted to a pathway analysis using ConsensusPathDB. The four α-diketones showed very similar results with regard to the number of activated and shared pathways. Overall, the number of signaling pathways decreased from α-to ß-to γ-diketones. Additionally, we reconstructed networks of genes that interact with one another and are associated with different adverse outcomes such as fibrosis, inflammation or apoptosis using the TRANSPATH-database. Transcription factor enrichment and upstream analyses with the geneXplain platform revealed highly interacting gene products (called master regulators, MRs) per case study compound. The mapping of the resultant MRs on the reconstructed networks, visualized similar gene regulation with regard to fibrosis, inflammation and apoptosis. This analysis showed that transcriptome data can strengthen the similarity assessment of compounds, which is of particular importance, e.g., in read-across approaches. It is one important step towards grouping of compounds based on biological profiles.

Epigallocatechin-3-gallate PEGylated poly(lactic-co-glycolic) acid nanoparticles mitigate striatal pathology and motor deficits in 3-nitropropionic acid intoxicated mice.

Aim: To compare free and nanoparticle (NP)-encapsulated epigallocatechin-3-gallate (EGCG) for the treatment of Huntington's disease (HD)-like symptoms in mice. Materials & methods: EGCG was incorporated into PEGylated poly(lactic-co-glycolic) acid NPs with ascorbic acid (AA). HD-like striatal lesions and motor deficit were induced in mice by 3-nitropropionic acid-intoxication. EGCG and EGCG/AA NPs were co-administered and behavioral motor assessments and striatal histology performed after 5 days. Results: EGCG/AA NPs were significantly more effective than free EGCG in reducing motor disturbances and depression-like behavior associated with 3-nitropropionic acid toxicity. EGCG/AA NPs treatment also mitigated neuroinflammation and prevented neuronal loss. Conclusion: NP encapsulation enhances therapeutic robustness of EGCG in this model of HD symptomatology. Together with our previous findings, this highlights the potential of EGCG/AA NPs in the symptomatic treatment of neurodegenerative diseases.

Dual-drug loaded nanoparticles of Epigallocatechin-3-gallate (EGCG)/Ascorbic acid enhance therapeutic efficacy of EGCG in a APPswe/PS1dE9 Alzheimer's disease mice model.

Epigallocatechin-3-gallate (EGCG) is a candidate for treatment of Alzheimer's disease (AD) but its inherent instability limits bioavailability and effectiveness. We found that EGCG displayed increased stability when formulated as dual-drug loaded PEGylated PLGA nanoparticles (EGCG/AA NPs). Oral administration of EGCG/AA NPs in mice resulted in EGCG accumulation in all major organs, including the brain. Pharmacokinetic comparison of plasma and brain accumulation following oral administration of free or EGCG/AA NPs showed that, whilst in both cases initial EGCG concentrations were similar, long-term (5-25 h) concentrations were ca. 5 fold higher with EGCG/AA NPs. No evidence was found that EGCG/AA NPs utilised a specific pathway across the blood-brain barrier (BBB). However, EGCG, empty NPs and EGCG/AA NPs all induced tight junction disruption and opened the BBB in vitro and ex vivo. Oral treatment of APPswe/PS1dE9 (APP/PS1) mice, a familial model of AD, with EGCG/AA NPs resulted in a marked increase in synapses, as judged by synaptophysin (SYP) expression, and reduction of neuroinflammation as well as amyloid ß (Aß) plaque burden and cortical levels of soluble and insoluble Aß(1-42) peptide. These morphological changes were accompanied by significantly enhanced spatial learning and memory. Mechanistically, we propose that stabilisation of EGCG in NPs complexes and a destabilized BBB led to higher therapeutic EGCG concentrations in the brain. Thus EGCG/AA NPs have the potential to be developed as a safe and strategy for the treatment of AD.

Comparison of migration disturbance potency of epigallocatechin gallate (EGCG) synthetic analogs and EGCG PEGylated PLGA nanoparticles in rat neurospheres.

Epigallocatechin gallate (EGCG), the main catechin of green tea, is described to have potential health benefits in several fields like oncology, neurology or cardiology. Currently, it is also under pre-clinical investigation as a potential therapeutic or preventive treatment during pregnancy against developmental adverse effects induced by toxic substances. However, the safety of EGCG during pregnancy is unclear due to its proven adverse effects on neural progenitor cells' (NPCs) migration. As lately several strategies have arisen to generate new therapeutic agents derived from EGCG, we have used the rat 'Neurosphere Assay' to characterize and compare the effects of EGCG structurally related compounds and EGCG PEGylated PLGA nanoparticles on a neurodevelopmental key event: NPCs migration. Compounds structurally-related to EGCG induce the same pattern of NPCs migration alterations (decreased migration distance, decreased formation of migration corona, chaotic orientation of cellular processes and decreased migration of neurons at higher concentrations). The potency of the compounds does not depend on the number of galloyl groups, and small structure variations can imply large potency differences. Due to their lower toxicity observed in vitro in NPCs, 4,4'-bis[(3,4,5-trihydroxybenzoyl)oxy]-1,1'-biphenyl and EGCG PEGylated PLGA nanoparticles are suggested as potential future therapeutic or preventive alternatives to EGCG during prenatal period.