Chemoproteomic Profiling Reveals 6PPDQ-Mediated Neurotoxicity in Rainbow Trout through Disruption of the Glutamate–Glutamine Cycle

6PPDQ, an emerging environmental pollutant derived from tire wear, has gained widespread attention due to its high acute toxicity toward salmonids, such as rainbow trout. It has been found to induce neurotoxic effects at environmentally relevant concentrations; yet, the underlying mechanisms remain elusive. Moreover, methods for the specific identification of 6PPDQ-interacting proteins are still lacking. Herein, we developed a clickable probe (6PPDQ-yne) and confirmed that it induces neurotoxic effects in rainbow trout similar to those caused by 6PPDQ through an acute lethality assay, neurobehavioral experiments, and histopathological analysis. We then employed the clickable probe to perform unbiased compound-centric chemical proteomics (CCCP) and systematically deconvoluted 6PPDQ target proteins in rainbow trout brains. The CCCP analysis identified 2,502 high-confidence target proteins, of which GS was selected for further investigation. Our results demonstrated that 6PPDQ directly binds to GS and downregulates its enzymatic activity. In fish brains, the inhibition of GS activity by 6PPDQ disrupted the glutamate-glutamine cycle and caused glutamate accumulation, which induced a massive calcium ion influx and ultimately triggered cell death. In summary, our study validates a critical protein target, laying a foundation for elucidating 6PPDQ-induced neurotoxicity mechanisms and providing a systematic paradigm for deconvoluting the toxicity mechanisms of emerging pollutants.