Chlorogenic acid counteracts mycophenolate mofetil-induced neurotoxicity in zebrafish and PC12 cells: A potential bioremediation strategy for aquatic pharmaceuticals

Pharmaceutical pollution, particularly from immunosuppressants like mycophenolate mofetil (MMF), has emerged as a significant environmental contaminant in aquatic ecosystems, frequently detected in wastewater at concentrations of 0.1-2.5 μg/L with an occurrence rate exceeding 80 %. This study demonstrates that chlorogenic acid (CGA), a plant-derived polyphenol found in coffee, fruits, and medicinal plants, effectively counters MMF-induced neurotoxicity in both zebrafish and neuronal cells. Specifically, CGA significantly protected PC12 cells from H₂O₂-induced damage by promoting cell proliferation, inhibiting apoptosis, and reducing oxidative stress in a dose-dependent manner. Exposure to 1 µM MMF reduced PC12 cell proliferation to 46.1 %, increased ROS 2.8-fold, elevated MDA 4.6-fold, and decreased SOD by 40 %. In zebrafish, motor-neuron axonal length was shortened to 56.6 % of control. Co-treatment with 10 µM CGA restored PC12 proliferation to 74.7 %, normalized ROS and MDA levels, and recovered SOD to 89 %. In zebrafish larvae, 100 µM CGA can extend the MMF-induced shortening of motor neuron axon length from 56.6 % to 89.5 %. Network pharmacology and ESR1 knockdown studies identified estrogen receptor 1 (ESR1) as a critical mediator of CGA's protective effects, supported by surface plasmon resonance (SPR) data confirming direct binding between CGA and ESR1. Notably, ESR1 silencing mitigated MMF-induced neurotoxicity, restoring PC12 cell proliferation to 64.8 % and zebrafish motor-neuron axonal length to 86 % of control. Our findings not only uncover the previously unrecognized environmental neurotoxicity of MMF but also elucidate CGA's multimodal protective mechanisms via ESR1 modulation. Furthermore, this work highlights the potential of dietary polyphenols as sustainable, eco-friendly agents for bioremediating pharmaceutical pollutants. By providing mechanistic insights into detoxification strategies, our study advances nature-based solutions for mitigating immunosuppressant contamination in aquatic systems, offering a promising approach to address emerging environmental challenges.