Suciu, Ilinca
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G × E interactions as a basis for toxicological uncertainty
To transfer toxicological findings from model systems, e.g. animals, to humans, standardized safety factors are applied to account for intra-species and inter-species variabilities. An alternative approach would be to measure and model the actual compound-specific uncertainties. This biological concept assumes that all observed toxicities depend not only on the exposure situation (environment = E), but also on the genetic (G) background of the model (G × E). As a quantitative discipline, toxicology needs to move beyond merely qualitative G × E concepts. Research programs are required that determine the major biological variabilities affecting toxicity and categorize their relative weights and contributions. In a complementary approach, detailed case studies need to explore the role of genetic backgrounds in the adverse effects of defined chemicals. In addition, current understanding of the selection and propagation of adverse outcome pathways (AOP) in different biological environments is very limited. To improve understanding, a particular focus is required on modulatory and counter-regulatory steps. For quantitative approaches to address uncertainties, the concept of “genetic” influence needs a more precise definition. What is usually meant by this term in the context of G × E are the protein functions encoded by the genes. Besides the g ene sequence, the regulation of the gene expression and function should also be accounted for. The widened concept of past and present “ g ene expression” influences is summarized here as G e . Also, the concept of “environment” needs some re-consideration in situations where exposure timing (E t ) is pivotal: prolonged or repeated exposure to the insult (chemical, physical, life style) affects G e . This implies that it changes the model system. The interaction of G e with E t might be denoted as G e × E t . We provide here general explanations and specific examples for this concept and show how it could be applied in the context of New Approach Methodologies (NAM).
Specific Attenuation of Purinergic Signaling during Bortezomib-Induced Peripheral Neuropathy In Vitro
Human peripheral neuropathies are poorly understood, and the availability of experimental models limits further research. The PeriTox test uses immature dorsal root ganglia (DRG)-like neurons, derived from induced pluripotent stem cells (iPSC), to assess cell death and neurite damage. Here, we explored the suitability of matured peripheral neuron cultures for the detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A two-step differentiation protocol, involving the transient expression of ectopic neurogenin-1 (NGN1) allowed for the generation of homogeneous cultures of sensory neurons. After >38 days of differentiation, they showed a robust response (Ca2+-signaling) to the P2X3 ligand α,β-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at ≥5 nM, while 50–200 nM was required in the PeriTox test to identify neurite damage and cell death. A 24 h treatment with low nM concentrations of bortezomib led to moderate increases in resting cell intracellular Ca2+ concentration but signaling through transient receptor potential V1 (TRPV1) receptors or depolarization-triggered Ca2+ influx remained unaffected. We interpreted the specific attenuation of purinergic signaling as a functional cell stress response. A reorganization of tubulin to form dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomicin, and MG-132 showed similar stress responses. Thus, the model presented here may be used for the profiling of new proteasome inhibitors in regard to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signaling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons.
A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling
Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.
Examination of microcystin neurotoxicity using central and peripheral human neurons
Microcystins (MC) are a group of cyanobacterial toxins that comprises MC-LF and other cyclic heptapeptides, best known as potent hepatotoxicants. Cell culture and epidemiological studies suggest that MC might also affect the nervous system, when there is systemic exposure e.g. via drinking water or food. We asked whether in vitro studies with human neurons could provide estimates on the neurotoxicity hazard of MC-LF. First, we used LUHMES neurons, a well-established test system for neurotoxicants and neuropathological processes. These central nervous system cells expressed OATP1A2, a presumed carrier of MC-LF, and we observed selective neurite toxicity in the µM range (EC20 = 3.3 µM ≈ 3.3 µg/ml). Toxicity paralleled transcriptome changes pointed towards attenuated cell maintenance and biosynthetic processes. Prolonged exposure for up to four days did not increase toxicity. As a second model, we used human dorsal root ganglia-like neurons. These peripheral nervous system cells represent parts of the nervous system not protected by the blood brain barrier in humans. Toxicity was observed in a similar concentration range (EC20 = 7.4 µM). We conclude that MC-LF poses a potential neurotoxic hazard in humans. The adverse effect concentrations observed here were orders of magnitude higher than those presumed to be encountered after normal nutritional or environmental exposure. However, the low µM concentrations found to be toxic are close to levels that may be reached after very excessive algae supplement intake.
Dynamic Metabolic and Transcriptional Responses of Proteasome-Inhibited Neurons
Proteasome inhibition is associated with parkinsonian pathology in vivo and degeneration of dopaminergic neurons in vitro. We explored here the metabolome (386 metabolites) and transcriptome (3257 transcripts) regulations of human LUHMES neurons, following exposure to MG-132 [100 nM]. This proteasome inhibitor killed cells within 24 h but did not reduce viability for 12 h. Overall, 206 metabolites were changed in live neurons. The early (3 h) metabolome changes suggested a compromised energy metabolism. For instance, AMP, NADH and lactate were up-regulated, while glycolytic and citric acid cycle intermediates were down-regulated. At later time points, glutathione-related metabolites were up-regulated, most likely by an early oxidative stress response and activation of NRF2/ATF4 target genes. The transcriptome pattern confirmed proteostatic stress (fast up-regulation of proteasome subunits) and also suggested the progressive activation of additional stress response pathways. The early ones (e.g., HIF-1, NF-kB, HSF-1) can be considered a cytoprotective cellular counter-regulation, which maintained cell viability. For instance, a very strong up-regulation of AIFM2 (=FSP1) may have prevented fast ferroptotic death. For most of the initial period, a definite life–death decision was not taken, as neurons could be rescued for at least 10 h after the start of proteasome inhibition. Late responses involved p53 activation and catabolic processes such as a loss of pyrimidine synthesis intermediates. We interpret this as a phase of co-occurrence of protective and maladaptive cellular changes. Altogether, this combined metabolomics–transcriptomics analysis informs on responses triggered in neurons by proteasome dysfunction that may be targeted by novel therapeutic intervention in Parkinson’s disease.
Distinct and Dynamic Transcriptome Adaptations of iPSC-Generated Astrocytes after Cytokine Stimulation
Astrocytes (ACs) do not only play a role in normal neurogenesis and brain homeostasis, but also in inflammatory and neurodevelopmental disorders. We studied here the different patterns of inflammatory activation triggered by cytokines in human induced pluripotent stem cell (iPSC)-derived ACs. An optimized differentiation protocol provided non-inflamed ACs. These cells reacted to TNFα with a rapid translocation of NFκB, while AC precursors showed little response. Transcriptome changes were quantified at seven time points (2-72 h) after stimulation with TNFα, IFNγ or TNFα plus IFNγ. TNFα triggered a strong response within 2 h. It peaked from 12-24 h and reverted towards the ground state after 72 h. Activation by IFNγ was also rapid, but the response pattern differed from that of TNFα. For instance, several chemokines up-regulated by TNFα were not affected by IFNγ. Instead, MHC-II-related antigen presentation was drastically enhanced. The combination of the two cytokines led to a stronger and more persistent response. For instance, TRIB3 up-regulation by the combination of TNFα plus IFNγ may have slowed NFκB inactivation. Additionally, highly synergistic regulation was observed for inflammation modifiers, such as CASP4, and for STAT1-controlled genes. The combination of the cytokines also increased oxidative stress markers (e.g., CHAC1), led to phenotypic changes in ACs and triggered markers related to cell death. In summary, these data demonstrate that there is a large bandwidth of pro-inflammatory AC states, and that single markers are not suitable to describe AC activation or their modulation in disease, development and therapy.
A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling
Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.
Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions
In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.
Functional alterations by a subgroup of neonicotinoid pesticides in human dopaminergic neurons
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.
Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors
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.