Fig 1.
Ferroptosis cell rescue activity, 15-lipoxygenase enzyme assay data, ORAC and RTA values for 18 vitamin E vitamers and metabolites.
Summary of cellular and enzyme assay potencies. Q7 ferroptosis cell rescue, 15-lipoxygenase (15-LO) enzyme inhibition, oxygen radical absorbance capacity (ORAC) values and the radical-trapping antioxidant (RTA) activity assays were measured for αT, its selected metabolites, and the non-metabolizable αTCC.
Fig 2.
The anti-ferroptotic activity of vitamin E and its metabolites.
(A) Ferroptosis was induced in Q7 cells by RSL3 treatment (2 μM 18 h), as measured by CellTiter-Glo® 2.0 ATP assay. The dose-dependent cytoprotective activity of vitamin E (αT), its metabolites (αTQ, αTHQ), or the synthetic carbochroman (αTCC) was assessed by co-treatment (in 24-point titration) with RSL3. Curves are representative of ≥9 independent experiments, of which 7 evaluated αTCC to a top concentration of 30 μM. (B) Ferroptosis was initiated in BODIPYTM 581/591 C11-prelabeled Q7 cells by RSL3 (2 μM) treatment, with cellular lipid oxidation assessed by the rate of change in green cellular fluorescence using time-lapse microscopy. Rescue compounds were co-treated (in 6-point titration) with RSL3. Mean ± SEM shown, (n = 6 to 9 technical replicates for each condition. Results are representative of 2 independent experiments.
Fig 3.
In vivo conversion of α-tocopherol to its quinone metabolite and quantification of intracellular concentrations of vitamin E and its metabolites in cells in vitro.
(A) To assess the in vivo conversion of αT to its quinone metabolite, stable deuterium-labelled α-tocopherol (d4-αT) was dosed orally to Sprague-Dawley male rats. Four hours after the final dose, plasma and tissues were collected for bioanalytical quantification of d4-αT and d4-αTQ by LC-MS/MS. While <1% of deuterium-labeled αT was detected as the quinone in the plasma fraction, varying levels of quinone conversion were observed in the tissues assessed, ranging from ~2% (liver) to ~50% (small intestine). (B) Vitamin E (αT) and its metabolites, vitamin E quinone (αTQ) and vitamin E hydroquinone (αTHQ), were detected simultaneously in Q7 striatal cells under basal growth conditions or supplementation with αT or αTQ (10 μM, 24 h). Results displayed are mean ± SD, n = 6; results from 1 experiment representative of 3 similar experiments. (C) Stacked bar graphs showing the mean proportions of αT, αTQ, and αTHQ quantified simultaneously under basal, αT- or αTQ-supplemented conditions using the succinate capping methodology described in Methods.
Fig 4.
Oxygen radical absorbance capacity (ORAC) and radical-trapping antioxidant (RTA) activity assays with αT, αTCC, αTQ, αTHQ in comparison with their ferroptosis rescue potency.
(A) ORAC activity of each compound compared to Trolox. Mean ± SEM (N = 6) displayed. (B) Overlay of kinetics of RTA activity of each compound (50 μM) prepared as 2 mol% in DOPC liposomes, detected through quenching of BODIPY™ 581/591 C11 (0.5 μM) emission at 600 nm. Mean ± SD (N = 3) displayed. (C) Comparison of RTA BODIPY quenching area-under-the-curve (AUC) of each compound (relative to the Control group, defined as 1) and pIC50 values for inhibition of RSL3-induced cellular BODIPY oxidation. Mean + SEM displayed; N = 3 for RTA assay, N = 6 for cell BODIPY assay.
Fig 5.
Lipid changes quantitated in RSL3-treated Q7 cells or their culture medium.
(A) RSL3-Q7 treated cells have elevated cellular levels of a lyso-PE-palmitoyl species (*p<0.05, Unpaired t-test with Welch’s correction). (B, C) RSL3-treated Q7 cells have elevated cellular and conditioned media levels of 15-HETE, an effect prevented by αTQ (5 μM) co-treatment (*p<0.05, **p<0.01, ***p<0.001, Shapiro-Welk normality test followed by ANOVA with Sidak’s multiple comparisons test or Kruskal-Wallis test with Dunn’s multiple comparisons test, respectively). (D, E) 12-HETE levels were not altered by RSL3 or αTQ treatment (ns, p>0.05, Shapiro-Welk normality test followed by Kruskal-Wallis test with multiple comparisons test. (F, G) Conditioned medium of RSL3-treated Q7 cells has elevated AA, but cellular levels are not significantly changed (***p<0.001, Shapiro-Welk normality test followed by ANOVA with Sidak’s multiple comparisons test).
Fig 6.
Inhibitory activity of 15-LO by vitamin E and its metabolites in an isolated enzyme assay and in Q7 cells.
(A) αTQ dose-dependently prevents RSL3-induced cellular release of 15-HETE (IC50 ~0.6 μM). Mean ± SD shown (n = 3), representative of 2 independent experiments. (B) αTHQ inhibited rabbit 15-lipoxygenase (15-LO) enzyme activity (IC50 2.45 μM) utilizing arachidonic acid as substrate, whereas αT, αTQ, and αTCC did not (up to 100 μM). Data are mean ± SD (n = 4, from 2 independent experiments). (C) EPR spectroscopy of soybean 15-lipoxygenase (SLO) with αTHQ or αT3HQ. Overlaid spectra focusing on the active site Fe3+ (g = 6.1) and the exogenous rhombic Fe3+ that is typically present in purified protein fractions (g = 4.3). Quantification of the Fe3+-SLO before treatment, after oxidation by lipid-hydroperoxide (set to 100%), and after compound treatment.
Fig 7.
Proposed target and mechanism of action of vitamin E.
The anti-ferroptotic effect of vitamin E results from a four-step biochemical mechanism: 1) oxidative hydrolysis of alpha-tocopherol (αT) to alpha-tocopherol quinone (αTQ); 2) reduction of alpha-tocopherol quinone (αTQ) to alpha-tocopherol hydroquinone (αTHQ); 3) inhibition of 15-LO via reduction of its non-heme Fe3+ center to the inactive Fe2+ state by alpha-tocopherol hydroquinone (αTHQ); and 4) inhibition of the ferroptosis cascade by blocking formation of lipid peroxidation products: polyunsaturated fatty acids (PUFA), hydroperoxidated polyunsaturated fatty acid (PUFA-OOH) and hydroxylated polyunsaturated fatty acid (PUFA-OH). The non-metabolizable isosteric analog of vitamin E αTCC retains its antioxidant activity, but does not inhibit ferroptosis.