Midazolam-induced developmental toxicity and neurotoxicity in zebrafish embryos: Insights from phenotypic, behavioural, and transcriptomic analyses.

Midazolam (MDZ), a widely used short-acting benzodiazepine, has been linked to developmental and neurotoxic effects, yet its underlying molecular mechanisms during early development remain unclear. This study examined the impact of MDZ on zebrafish embryos and larvae through phenotypic, behavioural, and transcriptomic analyses. Embryos were exposed to MDZ at 2 and 4 mg/L from 6 to 96 h post-fertilisation (hpf). MDZ exposure significantly increased mortality, delayed hatching, and induced morphological abnormalities, indicating developmental toxicity. Neurotoxic effects included elevated apoptosis in both brain and heart regions, reduced spontaneous movement, and altered motor responses to light-dark stimulation. MDZ also decreased heart rate and body size, highlighting its detrimental impact on growth and cardiac function. Transcriptomic profiling identified 1496 DEGs (380 up-regulated and 1116 down-regulated) and revealed altered expression of genes involved in GABAergic synapse, PPAR signalling, cardiac muscle contraction, oestrogen and MAPK signalling, apoptosis, phototransduction, and osteoclast differentiation. These molecular changes likely underpin the observed developmental and neurotoxic outcomes. Notably, locomotor impairments persisted up to 5 days post-fertilisation (dpf), partially recovered by 7 dpf, and approached normalization by 10 dpf. Collectively, these findings underscore the developmental risks of MDZ exposure and provide molecular insights that may inform safer clinical practice.