<italic>t</italic>-BuOOH induces ferroptosis in human and murine cell lines.

Reactive oxygen species (ROS)-induced apoptosis has been extensively studied. Increasing evidence suggests that ROS, for instance, induced by hydrogen peroxide (H₂O₂), might also trigger regulated necrotic cell death pathways. Almost nothing is known about the cell death pathways triggered by <italic>tertiary</italic>-butyl hydroperoxide (<italic>t</italic>-BuOOH), a widely used inducer of oxidative stress. The lipid peroxidation products induced by <italic>t</italic>-BuOOH are involved in the pathophysiology of many diseases, such as cancer, cardiovascular diseases, or diabetes. In this study, we exposed murine fibroblasts (NIH3T3) or human keratinocytes (HaCaT) to <italic>t</italic>-BuOOH (50 or 200 μM, respectively) which induced a rapid necrotic cell death. Well-established regulators of cell death, i.e., p53, poly(ADP)ribose polymerase-1 (PARP-1), the stress kinases p38 and c-Jun N-terminal-kinases 1/2 (JNK1/2), or receptor-interacting serine/threonine protein kinase 1 (RIPK1) and 3 (RIPK3), were not required for <italic>t</italic>-BuOOH-mediated cell death. Using the selective inhibitors ferrostatin-1 (1 μM) and liproxstatin-1 (1 μM), we identified ferroptosis, a recently discovered cell death mechanism dependent on iron and lipid peroxidation, as the main cell death pathway. Accordingly, <italic>t</italic>-BuOOH exposure resulted in a ferrostatin-1- and liproxstatin-1-sensitive increase in lipid peroxidation and cytosolic ROS. Ferroptosis was executed independently from other <italic>t</italic>-BuOOH-mediated cellular damages, i.e., loss of mitochondrial membrane potential, DNA double-strand breaks, or replication block. H₂O₂ did not cause ferroptosis at equitoxic concentrations (300 μM) and induced a (1) lower and (2) ferrostatin-1- or liproxstatin-1-insensitive increase in lipid peroxidation. We identify that <italic>t</italic>-BuOOH and H₂O₂ produce a different pattern of lipid peroxidation, thereby leading to different cell death pathways and present <italic>t</italic>-BuOOH as a novel inducer of ferroptosis. [ABSTRACT FROM AUTHOR]