Drospirenone inhibits neurobehavioral activity in zebrafish larvae by modulating NMDA receptors

Progestogens, primarily used in assisted reproduction and tocolytic therapy, have emerged as environmental pollutants due to their widespread use in medicine and aquaculture. Their release into various ecosystems has raised concerns about their potential neurodevelopmental effects. Drospirenone (DRS), an extensively used synthetic progestin, was identified in bioinformatic analysis as a potential disruptor of NMDA receptors (NMDARs), crucial for neuronal signaling. To investigate this, zebrafish embryos were exposed to DRS from 2 h post-fertilization (hpf) to 14 days post-fertilization (dpf). Behavioral assays revealed that DRS exposure impaired locomotor activity, reduced anxiety-like responses, and diminished social behaviors. Quantitative PCR and immunofluorescence staining showed that DRS disrupted the expression of several NMDAR subunits, suppressed apoptosis, and promoted neuronal proliferation in the embryonic brain. Co-exposure with NMDA, an NMDAR agonist, reversed the neurobehavioral deficits, normalized NMDARs expression, and restored apoptosis-proliferation balance. Molecular docking, DARTS assays, and downstream marker measurements indicated target engagement between DRS and the Grin1 and Grin2 subunits of NMDAR, suggesting that DRS may alter receptor conformation and function. In conclusion, DRS induces significant neurodevelopmental toxicity in zebrafish larvae, likely by suppressing NMDAR activity and disrupting neuronal apoptosis and proliferation. These findings highlight potential risks of DRS to aquatic life and human health.