Revisiting Quinone-Induced Oxidative Stress via Structure-Related Protein Alkylation

Quinones and transition metals are two major chemical components in particulate matter (PM) driving oxidative potentials, but their relative contributions are inconsistent between acellular and cellular assays. In this study, we examined the structure-related oxidative potentials of 8 quinones and 6 transition metals, using both acellular (DTT) and cellular (Nrf2) assays. Transition metals showed high DTT but low Nrf2 responses, while quinones displayed strong Nrf2 responses and moderate DTT responses, indicating that quinones can induce cellular oxidative stress via additional ROS-independent pathways. To explore the molecular mechanism of quinones inducing oxidative stress in depth, we used free cysteine and direct-infusion mass spectrometry to reveal a strong correlation between the thiol reactivities of 8 quinones and their Nrf2 responses. Furthermore, activity-based protein profiling (ABPP) confirmed that quinone-induced protein alkylation in cell lysates is determined by their structure-related thiol reactivities. Notably, we observed an inverted U-shaped trend between quinone-thiol reactivity and their Nrf2 responses. Live-cell ABPP revealed that highly reactive benzoquinone is sequestered by media proteins before entering live cells, which likely accounts for the inverted U-shape toxicity trend. This study highlights the importance of protein alkylation by quinones, a pathway previously underestimated by the acellular DTT assay.