Ückert, Anna-Katharina
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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.
P14-16 Application of various human in vitro systems for the confirmation of cellular targets and the molecular toxicity of glutathione conjugates of Trichloroethylene
EFSA Pilot Project on New Approach Methodologies (NAMs) for Tebufenpyrad Risk Assessment : Part 2. Hazard characterisation and identification of the Reference Point
This Project was performed to provide an in vitro Point of Departure (PoD) for an Adverse Outcome Pathway (AOP)-informed Integrated Approach to Testing and Assessment (IATA), concerning a potential risk of parkinsonian motor deficits after long-term exposure to Tebufenpyrad. The AOP considered was AOP:3. Assays were performed for Key Event (KE) 1, KE2 (mitochondrial dysfunction) and KE4 of the AOP, based on the use of human dopaminergic neurons (LUHMES cells). KE1 (inhibition of complex I of the mitochondrial respiratory chain) was considered equivalent to the molecular initiating event (MIE). KE4 (dopaminergic cell degeneration) was considered as alternative AO (target cell degeneration). All necessary and linear steps of the AOP (i.e. KE2,3,4) were investigated by experimental test methods. The three major objectives were: (i) generation of potential PoD data from each assay; (ii) evaluation and optimization of the consistency of these data; (iii) selection of a PoD to be used for further risk assessment. During the optimization phase, assay conditions that reflect brain metabolism (MitoMet conditions) were implemented, and degeneration of neurites was considered the most relevant neuropathological endpoint. With this setting, the potential PoD ranged from 6 nM to 45 nM. The selection of the PoD put emphasis on more chronic effects and on the assay closest to the AO. Thus the KE4 assay, measuring neurite degeneration under MitoMet conditions was chosen, and the PoD was 8 nM. This value has a confidence interval of about one log10-fold change from the lowest to the highest estimate (range: 3 nM – 30 nM), based on the experimental variations we observed. Some biokinetics measurements were undertaken to help estimating cell concentrations. These data had a high uncertainty concerning the measurements and the model assumptions. Based on the assumptions we consider most realistic and robust, we suggest a cell/tissue (brain) concentration of 8-40 nM to be associated with a potential onset of toxicity of Tebufenpyrad.
Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models
Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.