Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery

Klose, Jördis; Pahl, Melanie; Bartmann, Kristina; Bendt, Farina; Blum, John D.; Dolde, Xenia; Förster, Nils; Holzer, Anna‐Katharina; Hübenthal, Ulrike; Keßel, Hagen Eike; Koch, Katharina; Masjosthusmann, Stefan; Schneider, Sabine; Stürzl, Lynn‐Christin; Woeste, Selina; Rossi, Andrea; Behl, Mamta; Leist, Marcel; Tigges, Julia; Fritsche, Ellen

doi:10.1007/s10565-021-09603-2

Cited by 38 publications

(49 citation statements)

References 126 publications

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“…The PeriTox test is a well established in vitro screening assay using human iPSC-derived peripheral neurons to identify peripheral neurotoxicants [28]. It has been successfully used to identify environmental neurotoxicants by assessing their effects on the neurite structures [40][41][42]. However, for pre-clinical drug testing, an increased sensitivity in detecting the potential neurotoxicity of chemotherapeutics is desirable.…”

Section: Discussionmentioning

confidence: 99%

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Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

Holzer

Suciu

Karreman

et al. 2022

Preprint

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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 detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A 2-step differentiation protocol, involving 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+-signalling) to the P2X3 ligand α,β-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at ≥ 5 nM, while 50-200 nM were 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+], but signalling through transient receptor potential-V1 (TRPV1) receptors or depolarization-triggered Ca2+-influx remained unaffected. We interpret the specific attenuation of purinergic signalling as functional cell stress response. A reorganization of tubulin to dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomycin and MG-132 showed similar stress responses. Thus, the model presented here may be used for profiling of new proteasome inhibitors as to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signalling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons.

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Section: Discussionmentioning

confidence: 99%

“…In order to test, whether also functional properties were similar, we investigated toxicant-sensitivity. The PeriTox test, a well established screening assay [40][41][42], was used to assess effects on the neurite area and the cell viability of immature neurons on DoD0.…”

Section: Human Ipsc-derived Peripheral Neurons For Toxicity Testingmentioning

confidence: 99%

Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

Holzer

Suciu

Karreman

et al. 2022

Preprint

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“…Thus, we believe the argument that safety is ensured because typical human exposures are below the reference dose (when known) has not been demonstrated. Also important is that human exposures occur throughout lifetimes to complex mixtures of flame retardants and plasticizers, which may have additive or synergistic effects that are not accounted for in current regulatory assessments (Klose et al 2021). Such exposures are rapidly increasing on a global scale (Blum et al 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Other studies using a battery of cell-based and in vitro assays have reported compromised neurodifferentiation, neurite outgrowth, and electrical activity by numerous OPEs (Behl et al 2015;Hausherr et al 2014;Shafer et al 2019), sometimes at concentrations lower than for known neurotoxic OPPs (Dishaw et al 2014a;Ryan et al 2016). Furthermore, other key processes involved in neurodevelopment-such as proliferation, migration, and oligodendrocyte differentiation-have been shown to be affected by exposure to OPEs (Klose et al 2021).…”

Section: Nonmammalian and In Vitro Modelsmentioning

confidence: 99%

Beyond Cholinesterase Inhibition: Developmental Neurotoxicity of Organophosphate Ester Flame Retardants and Plasticizers

Patisaul

Behl

Birnbaum

et al. 2021

Environ Health Perspect

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Background: To date, the toxicity of organophosphate esters has primarily been studied regarding their use as pesticides and their effects on the neurotransmitter acetylcholinesterase (AChE). Currently, flame retardants and plasticizers are the two largest market segments for organophosphate esters and they are found in a wide variety of products, including electronics, building materials, vehicles, furniture, car seats, plastics, and textiles. As a result, organophosphate esters and their metabolites are routinely found in human urine, blood, placental tissue, and breast milk across the globe. It has been asserted that their neurological effects are minimal given that they do not act on AChE in precisely the same way as organophosphate ester pesticides. Objectives: This commentary describes research on the non-AChE neurodevelopmental toxicity of organophosphate esters used as flame retardants and plasticizers (OPEs). Studies in humans, mammalian, nonmammalian, and in vitro models are presented, and relevant neurodevelopmental pathways, including adverse outcome pathways, are described. By highlighting this scientific evidence, we hope to elevate the level of concern for widespread human exposure to these OPEs and to provide recommendations for how to better protect public health. Discussion: Collectively, the findings presented demonstrate that OPEs can alter neurodevelopmental processes by interfering with noncholinergic pathways at environmentally relevant doses. Application of a pathways framework indicates several specific mechanisms of action, including perturbation of glutamate and gamma-aminobutyric acid and disruption of the endocrine system. The effects may have implications for the development of cognitive and social skills in children. Our conclusion is that concern is warranted for the developmental neurotoxicity of OPE exposure. We thus describe important considerations for reducing harm and to provide recommendations for government and industry decision makers. https://doi.org/10.1289/EHP9285

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“…As cell source, we used NCCs differentiated from pluripotent stem cells. Such cells have been characterized by comprehensive transcriptome analysis, and they have been used successfully as test system for in vitro assays (Nyffeler et al, 2017a;Pallocca et al, 2017;Zimmer et al, 2014;Klose et al, 2021;Lee et al, 2010).…”

Section: Establishment Of a Chemotaxis Assay Based On Human Ncsmentioning

confidence: 99%

Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays

Dolde

Karreman

Wiechers

et al. 2021

Preprint

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Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chemotactic for NCCs in animal models (e.g. VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined in a broad screening approach, whether human cells would produce any factor able to trigger NCC migration. We found that HepG2 hepatoma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease-sensitivity suggested a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

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Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery

Cited by 38 publications

References 126 publications

Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

Beyond Cholinesterase Inhibition: Developmental Neurotoxicity of Organophosphate Ester Flame Retardants and Plasticizers

Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays

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