The central mechanism in ferroptosis linking lipid hydroperoxide accumulation with cell death remains poorly understood. Although lipid hydroperoxides are known to break down to reactive lipid-derived electrophiles (LDEs), the ability of cells to detoxify increasing LDE levels during ferroptosis has not been studied. Here, we developed an assay (ElectrophileQ) correlating the cellular retention vs. excretion of a fluorogenic lipophilic electrophile (AcroB) that enables live-cell assessment of the glutathione-mediated LDE conjugation and adduct export steps of the LDE detoxification pathway. This method revealed that during ferroptosis, LDE detoxification failure occurs through decreased conjugation or export impairment, amplifying cellular electrophile accumulation. Notably, ferroptosis susceptibility was increased following exacerbation of LDE-adduct export impairment through export channel inhibition. Our results expand understanding of the ferroptosis molecular cell death mechanism to position the LDE detoxification pathway as a ferroptosis-relevant therapeutic target. We envision the ElectrophileQ assay becoming an invaluable tool for studying ferroptosis and cellular health.
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