Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

Acrylamide alters neurotransmitter induced calcium responses in murine ESC-derived and primary neurons.

Stem cell-derived specialized cell types are of interest as an alternative cell system to identify and research neurotoxic effects and modes of action. Developmental toxicity may be studied during differentiation, while organ-specific toxicity may be assessed in fully functional cells, such as neurons. In this study we tested if fully differentiated neurons derived from murine embryonic stem cells (ESCN) could be used to investigate the effects of the well characterized neurotoxic model compound acrylamide (ACR) and if ESCN behave similar to murine primary cortical neurons (pCN) from 16 days old embryos. We characterized the differentiation process of cryopreserved ESC-derived neural precursor cells (NPC) differentiating to ESCN. During the differentiation process (days 11-20) a strong increase in calcium responses to glutamate, acetylcholine and GABA were observed. Moreover, neuron specific marker proteins, ß-III-tubulin, MAP2, Tau, Rbfox3 and synaptophysin showed similar patterns to pCN. In ESCN and pCN the neuronal structure, e.g. neurites, was not affected by low concentrations of ACR [0.5-1.6mM]. However, 24h incubation periods with 0.5-1.6mM ACR led to a reduction of acetylcholine and glutamate induced calcium responses. In conclusion, we show that non-cytotoxic concentrations of ACR alter neurotransmission in ESCN as well as pCN.

Assessment of chemical-induced impairment of human neurite outgrowth by multiparametric live cell imaging in high-density cultures.

Chemicals that specifically alter human neurite outgrowth pose a hazard for the development of the nervous system. The identification of such compounds remains a major challenge, especially in a human test system. To address this issue, we developed an imaging-based procedure in LUHMES human neuronal precursor cells to quantify neurite growth of unfixed cultures. Live imaging allowed the simultaneous evaluation of cell viability and neurite outgrowth within one culture dish. The procedure was used to test the hypothesis that inhibitors of specific pathways can impair neurite outgrowth without affecting cell viability. Although the cells were grown at high density to allow extensive networking, overall neurite growth in this complex culture was quantified with a signal-to-noise ratio of > 50. Compounds such as U0126 slowed the extension of neuronal processes at concentrations > 4 times lower than those causing cell death. High numbers of individual viable cells without neurites were identified under such conditions, and neurite outgrowth recovered after washout of the chemical. Also an extension-promoting compound, Y-27632, was identified by this unique multiparametric imaging approach. Finally, the actions of unspecific cytotoxicants such as menadione, cadmium chloride, and sodium dodecyl sulfate were tested to evaluate the specificity of the new assay. We always found a ratio of EC50 (cell death)/EC50 (neurites) < 4 for such chemicals. The described novel test system may thus be useful both for high-throughput screens to identify neuritotoxic agents and for their closer characterization concerning mode of action, compound interactions, or the reversibility of their effects.