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Sulindac, identified through a small clinical anti-inflammatory drug screening, elicited neurodevelopmental toxicity. a Flow diagram of a small-scale anti-inflammatory drug screening for brain developmental toxicity. Wild-type (WT) embryos were collected and arrayed in 24-well plates at 2 dpf. Each well contained the compound dissolved to 10 μM in DMSO and E3 embryo medium. Screening was conducted via acridine orange (AO) staining at 4 dpf. b–i Representative results of AO staining. Among the 46 anti-inflammatory drugs, only sulindac (c) induced apoptosis in the zebrafish midbrain (dashed line), while the other drugs, such as acetaminophen (d), aspirin (e), ibuprofen (f), naproxen (g), rofecoxib (h) and diclofenac (i), had no significant toxic effects compared to the DMSO (b). The experiments were independently repeated twice with similar results, and at least six zebrafish were observed each time. j AO+ cells (green signals) were colocalized with neuronal cells (red signals) in the midbrains of Tg(Xla.Tubb:DsRedx) embryos indicated by yellow arrows. k Quantification of the yellow double-positive cells in the midbrain. The values represent the means ± SDs (n = 4 larvae for per group, each dot denotes one larva). Statistics calculated by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
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