Differential Contributions of Distinct Free Radical Peroxidation Mechanisms to the Induction of Ferroptosis

[Image: see text] Ferroptosis is a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Lipid peroxidation can propagate through either the hydrogen-atom transfer (HAT) or peroxyl radical addition (PRA) mechanism. However, the contribution of the PRA mechanism to the induction of ferroptosis has not been studied. In this study, we aim to elucidate the relationship between the reactivity and mechanisms of lipid peroxidation and ferroptosis induction. We found that while some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins D(3) and A, and coenzyme Q10, suppress ferroptosis, both nonconjugated and conjugated PUFAs enhanced cell death induced by RSL3, a ferroptosis inducer. Importantly, we found that conjugated PUFAs, including conjugated linolenic acid (CLA 18:3) and conjugated linoleic acid (CLA 18:2), can induce or potentiate ferroptosis much more potently than nonconjugated PUFAs. We next sought to elucidate the mechanism underlying the different ferroptosis-inducing potency of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated and nonconjugated PUFAs are incorporated into distinct cellular lipid species. The different peroxidation mechanisms predict the formation of higher levels of reactive electrophilic aldehydes from conjugated PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping and mass spectrometry. RNA sequencing revealed that protein processing in the endoplasmic reticulum and proteasome are among the most significantly upregulated pathways in cells treated with CLA 18:3, suggesting increased ER stress and activation of unfolded protein response. These results suggest that protein damage by lipid electrophiles is a key step in ferroptosis.