Leist, Marcel
0000-0002-3778-8693 Berufsbeschreibung
Nachname
Vorname
Name
Suchergebnisse Publikationen
Novel Proline Transporter Inhibitor (LQFM215) Presents Antipsychotic Effect in Ketamine Model of Schizophrenia
The glutamatergic hypothesis of schizophrenia suggests a correlation between NMDA receptor hypofunction and negative psychotic symptoms. It has been observed that the expression of the proline transporter (PROT) in the central nervous system (CNS) is associated with glutamatergic neurotransmission, as l-proline has the capacity to activate and modulate AMPA and NMDA receptors. In this study, we aimed to investigate whether inhibition of proline transporters could enhance glutamatergic neurotransmission and potentially exhibit antipsychotic effects in an experimental schizophrenia model. Using molecular dynamics analysis in silico, we validated an innovative PROT inhibitor, LQFM215. We quantified the cytotoxicity of LQFM215 in the Lund human mesencephalic cell line (LUHMES). Subsequently, we employed the ketamine-induced psychosis model to evaluate the antipsychotic potential of the inhibitor, employing behavioral tests including open-field, three-chamber interaction, and prepulse inhibition (PPI). Our results demonstrate that LQFM215, at pharmacologically active concentrations, exhibited negligible neurotoxicity when astrocytes were co-cultured with neurons. In the ketamine-induced psychosis model, LQFM215 effectively reduced hyperlocomotion and enhanced social interaction in a three-chamber social approach task across all administered doses. Moreover, the compound successfully prevented the ketamine-induced disruption of sensorimotor gating in the PPI test at all tested doses. Overall, these findings suggest that PROT inhibition could serve as a potential therapeutic target for managing symptoms of schizophrenia model.
Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells
Animal studies for embryotoxicity evaluation of potential therapeutics and environmental factors are complex, costly, and time-consuming. Often, studies are not of human relevance because of species differences. In the present study, we recapitulated the process of cardiomyogenesis in human induced pluripotent stem cells (hiPSCs) by modulation of the Wnt signaling pathway to identify a key cardiomyogenesis gene signature that can be applied to identify compounds and/or stress factors compromising the cardiomyogenesis process. Among the 23 tested teratogens and 16 non-teratogens, we identified three retinoids including 13- cis -retinoic acid that completely block the process of cardiomyogenesis in hiPSCs. Moreover, we have identified an early gene signature consisting of 31 genes and associated biological processes that are severely affected by the retinoids. To predict the inhibitory potential of teratogens and non-teratogens in the process of cardiomyogenesis we established the “Developmental Cardiotoxicity Index” (CDI 31g ) that accurately differentiates teratogens and non-teratogens to do or do not affect the differentiation of hiPSCs to functional cardiomyocytes.
Deconvoluting gene and environment interactions to develop an “epigenetic score meter” of disease
Human health is determined both by genetics (G) and environment (E). This is clearly illustrated in groups of individuals who are exposed to the same environmental factor showing differential responses. A quantitative measure of the gene–environment interactions (GxE) effects has not been developed and in some instances, a clear consensus on the concept has not even been reached; for example, whether cancer is predominantly emerging from “bad luck” or “bad lifestyle” is still debated. In this article, we provide a panel of examples of GxE interaction as drivers of pathogenesis. We highlight how epigenetic regulations can represent a common connecting aspect of the molecular bases. Our argument converges on the concept that the GxE is recorded in the cellular epigenome, which might represent the key to deconvolute these multidimensional intricated layers of regulation. Developing a key to decode this epigenetic information would provide quantitative measures of disease risk. Analogously to the epigenetic clock introduced to estimate biological age, we provocatively propose the theoretical concept of an “epigenetic score‐meter” to estimate disease risk.
Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells
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.
Risk assessment of parabens in a transcriptomics-based in vitro test
Parabens have been used for decades as preservatives in food, drugs and cosmetics. The majority however, were banned in 2009 and 2014 leaving only methyl-, ethyl-, propyl-, and butyl-derivates available for subsequent use. Methyl- and propylparaben have been extensively tested in vivo, with no resulting evidence for developmental and reproductive toxicity (DART). In contrast, ethylparaben has not yet been tested for DART in animal experiments, and it is currently debated if additional animal studies are warranted. In order to perform a comparison of the four currently approved parabens, we used a previously established in vitro test based on human induced pluripotent stem cells (iPSC) that are exposed to test substances during their differentiation to neuroectodermal cells. EC50 values for cytotoxicity were 906 μM, 698 μM, 216 μM and 63 μM for methyl-, ethyl-, propyl- and butylparaben, respectively, demonstrating that cytotoxicity increases with increasing alkyl chain length. Genome-wide analysis demonstrated that FDR-adjusted significant gene expression changes occurred only at cytotoxic or close to cytotoxic concentrations, for example 1720 differentially expressed genes (DEG) at 1000 μM ethylparaben, 1 DEG at 316 μM, and no DEG at 100 μM or lower concentrations. The highest concentration of ethylparaben that did not induce any cytotoxicity nor DEG was 1670-fold above the highest published concentrations reported in biomonitoring studies (60 nM ethylparaben in cord blood). In conclusion, cytotoxicity and gene expression alterations of ethylparaben occurred at concentrations of approximately three orders of magnitude above human blood concentrations; moreover, the substance fitted well into a scenario where toxicity increases with the alkyl chain length, and gene expression changes only occur at cytotoxic or close to cytotoxic concentrations. Therefore, no evidence was obtained suggesting that in vivo DART with ethylparaben would lead to different results as the methyl- or propyl derivates.
Basic concepts of mixture toxicity and relevance for risk evaluation and regulation
Exposure to multiple substances is a challenge for risk evaluation. Currently, there is an ongoing debate if generic “mixture assessment/allocation factors” (MAF) should be introduced to increase public health protection. Here, we explore concepts of mixture toxicity and the potential influence of mixture regulation concepts for human health protection. Based on this analysis, we provide recommendations for research and risk assessment. One of the concepts of mixture toxicity is additivity. Substances may act additively by affecting the same molecular mechanism within a common target cell, for example, dioxin-like substances. In a second concept, an “enhancer substance” may act by increasing the target site concentration and aggravating the adverse effect of a “driver substance”. For both concepts, adequate risk management of individual substances can reliably prevent adverse effects to humans. Furthermore, we discuss the hypothesis that the large number of substances to which humans are exposed at very low and individually safe doses may interact to cause adverse effects. This commentary identifies knowledge gaps, such as the lack of a comprehensive overview of substances regulated under different silos, including food, environmentally and occupationally relevant substances, the absence of reliable human exposure data and the missing accessibility of ratios of current human exposure to threshold values, which are considered safe for individual substances. Moreover, a comprehensive overview of the molecular mechanisms and most susceptible target cells is required. We conclude that, currently, there is no scientific evidence supporting the need for a generic MAF. Rather, we recommend taking more specific measures, which focus on compounds with relatively small ratios between human exposure and doses, at which adverse effects can be expected.
Proteomic responses in the human dopaminergic LUHMES cell line to imidacloprid and its metabolites imidacloprid-olefin and desnitro-imidacloprid
Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitro-imidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10–100 μM) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 μM IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.
Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration
The causes of nigrostriatal cell death in idiopathic Parkinson’s disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the “basal slowing response” assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.
Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging
Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3β4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT).
New approach methodologies in human regulatory toxicology : Not if, but how and when!
The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of “New Approach Methodologies” (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure–activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today’s regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free “Next Generation Risk Assessment” (NGRA).