Developmental Neurotoxicity (DNT)

The use of human pluripotent stem cells offers the opportunity to investigate the potential effects of xenobiotics on the nervous central system during the early stage of development of embryos or in later life stages. During research and development, early information on the neurotoxicity or developmental toxicity of chemicals can guide the selection of the process for candidate products.

For the assessment of pesticides or drugs, in vitro safety testing is particularly useful for the elucidation of the mode of action.

For pharmaceutical applications, in vitro efficacy testing can be used in the selection of new drug candidates.

Services provided include:

  1. Use of in vitro tests for the assessing of developmental toxicity (DT) and developmental neurotoxicity (DNT) hazard.
  2. Use of in vitro tests for the assessing of neurotoxicity (NT) hazard
  3. Use of in vitro tests for assessing efficacy and mode of action (MoA) of neuroprotective drugs.
  4. Complementation of in vivo studies for NT, DNT, DT to provide important information on MoA of chemicals.

Descriptions of Assays

Our assays cover crucial functional endpoints for (developmental) neurotoxicity testing:

  • Assay to test viability and impairment of migration of human neural crest cells (cMINC; UKN2);
  • Assay to test compound-derived impairment in neurite outgrowth in human mature dopaminergic neurons (NeuriTox; UKN4);
  • Assay to test compound-derived impairment in neurite outgrowth in human iPSC-derived immature dorsal root ganglia (iDRG) neurons (PeriTox; UKN5).
  • Neurofunctional assays based on receptor interactions, for dopamine transporter and assessing network formation (e.g. assessment of Ca²+ signaling and electrical activity on multi-electrodes arrays);
  • Assays for neuroprotective drug candidates in conventional cell cultures and complex organoids.

Assay to test viability and impairment of migration of human neural crest cells (cMINC; UKN2).

This in vitro test method uses human neural crest cells (NCCs) generated from induced pluripotent stem cells (iPSC). It assesses disturbances of NCC function during fetal development. The number of migrated NCC, as well as cell viability are measured simultaneously using high content imaging. The data of this method are meant to predict developmental disorders and malformations e.g. neural tube defects or craniofacial malformations caused by compound exposure during fetal development. The method has a well-established prediction model, and it has been used in the screening of several medium-sized compound libraries.

Assay to test compound-derived impairment in neurite outgrowth in human mature dopaminergic neurons (NeuriTox; UKN4).

This in vitro test method is based on human neurons (LUHMES cells) at a stage of neurite growth (day 2 of differentiation). It assesses (a) disturbances in the development of the nervous system/brain structures, and (b) direct damage to the adult nervous system, by exposure to toxicants.

Variant 1: The neurite area (which serves as indirect measurement of neuronal interconnectivity) of stained differentiating neurons, as well as cellular viability are measured simultaneously at 24 h after toxicant exposure using high content imaging. The processes of neurite outgrowth and cell death are measured.

Variant 2: The neurite area (which serves as indirect measurement of neuronal interconnectivity) of stained differentiating neurons, as well as cellular viability are measured simultaneously at 24 h after toxicant exposure using high content imaging. The processes of neurite outgrowth and cell death are measured. Assessment is performed in the presence of either galactose or glucose. The ratios (glucose/galactose) are used to predict mitochondrial toxicity.

Variant 3: The proteasomal function of cells is assessed at 24 h after toxicant exposure by a fluorogenic substrate that increases in fluorescence when the proteasome is active.

Variant 4: The intracellular ATP level of the neurons is assessed at 24 h after toxicant exposure. It is assumed that dead cells do not contain ATP. Thus, the population ATP level is used as indirect measure of cell death. The rate of mitochondrial metabolism, measured by oxygen consumption, is assessed directly (within 15 minutes) after exposure.

The data of this method are meant to predict (a) developmental disorders caused by compound exposure during fetal development, and (b) damage to the developed nervous system, in particular to dopaminergic parts of the nervous system. The method has been used in the screening of medium-sized compound libraries, has undergone some mechanistic evaluation, and has been linked to AOP-3 (parkinsonian motor deficits).

Assay to test compound-derived impairment in neurite outgrowth in human iPSC-derived immature dorsal root ganglia (iDRG) neurons (PeriTox; UKN5).

This in vitro test method is based on human iPSC-derived immature dorsal root ganglia (iDRG) neurons at a stage of neurite growth. It assesses (a) disturbances in the development of the (peripheral) nervous system, and (b) direct damage to the peripheral nervous system, by exposure to toxicants. The neurite area (which serves as indirect measurement of neuronal interconnectivity) of stained differentiating neurons, as well as cellular viability are measured simultaneously using high content imaging. The processes of neurite outgrowth and cell death are measured. The data of this method are meant to predict (a) developmental disorders in children caused by compound exposure during fetal development, and (b) damage to the developed nervous system, in particular to the peripheral nervous system. The method predicts neurotoxicity. It has been used in the screening of medium-sized compound libraries, and has undergone some mechanistic evaluation, and been linked to AOP-279 (AOPwiki ID) / ETR09N (EU-ToxRisk AOP task ID) (Peripheral neuropathy caused by microtubule interacting drugs).

Method description is available online at https://eutoxrisk.edelweissconnect.com/public/

References
  • Delp J, Funke M, Rudolf F, et al. Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants. Arch Toxicol. 2019 Jun;93(6):1585-1608. doi: 10.1007/s00204-019-02473-y. Epub 2019 Jun 12. PMID: 31190196.
  • Delp J, Gutbier S, Klima S, et al. A high-throughput approach to identify specific neurotoxicants/ developmental toxicants in human neuronal cell function assays. ALTEX. 2018;35(2):235-253. doi: 10.14573/altex.1712182. Epub 2018 Jan 21. Erratum in: ALTEX. 2019;36(3):505. PMID: 29423527.
  • Dreser N, Madjar K, Holzer AK, et al. Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances. Arch Toxicol. 2020 Jan;94(1):151-171. doi: 10.1007/s00204-019-02612-5. Epub 2019 Nov 11. PMID: 31712839.
  • Gutbier S, Spreng AS, Delp J, et al. Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress. Cell Death Differ. 2018 Dec;25(12):2101-2117. doi: 10.1038/s41418-018-0229-x. Epub 2018 Nov 2. PMID: 30390092; PMCID: PMC6261954.
  • Klima S, Suciu I, Hoelting L, et al. Examination of microcystin neurotoxicity using central and peripheral human neurons. ALTEX. 2020 Jun 23. doi: 10.14573/altex.2003182. Epub ahead of print. PMID: 32591837.
  • Krebs A, Nyffeler J, Karreman C, Schmidt BZ, Kappenberg F, Mellert J, Pallocca G, Pastor M, Rahnenführer J, Leist M. Determination of benchmark concentrations and their statistical uncertainty for cytotoxicity test data and functional in vitro assays. ALTEX. 2020;37(1):155-163. doi: 10.14573/altex.1912021. Epub 2019 Dec 12. PMID: 31833558.
  • Krebs A, van Vugt-Lussenburg BMA, Waldmann T, et al. The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods. Arch Toxicol. 2020 Jul;94(7):2435-2461. doi: 10.1007/s00204-020-02802-6. Epub 2020 Jul 6. PMID: 32632539; PMCID: PMC7367925.
  • Nyffeler J, Dolde X, Krebs A, et al. Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library. Arch Toxicol. 2017 Nov;91(11):3613-3632. doi: 10.1007/s00204-017-1977-y. Epub 2017 May 5. PMID: 28477266.
  • Nyffeler J, Karreman C, Leisner H, et al. Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants. ALTEX. 2017;34(1):75-94. doi: 10.14573/altex.1605031. Epub 2016 Jul 27. PMID: 27463612.