Antioxidant-independent activities of alpha-tocopherol

Abstract

Alpha-tocopherol (vitamin E) is a plant-derived dietary lipid that is essential for the health of most animals, including humans. Originally discovered as a fertility factor in rodents, the primary health-promoting properties of the vitamin in humans was shown to be protection of neuromuscular functions. Heritable vitamin E deficiency manifests in spinocerebellar ataxia that can be stabilized by timely supplementation with high-dose α-tocopherol. The molecular basis for α-tocopherol's biological activities has been attributed primarily to the vitamin's efficacy in preventing lipid peroxidation in membranes and lipoproteins, but the possibility that the vitamin possesses additional biological activities has been postulated and debated in the literature without conclusive resolution. We designed and synthesized a novel analog of α-tocopherol, 6-hydroxymethyl α-tocopherol (6-HMTC), which retains most of the vitamin's structural, physical, and biochemical properties, yet lacks measurable radical-trapping antioxidant activity. 6-HMTC bound to the tocopherol transfer protein with high (nanomolar) affinity, like that of the natural vitamin, attesting to the analog's preservation of structural integrity. Yet, 6-HMTC did not inhibit lipid peroxidation or associated ferroptotic cell death. Notably, 6-HMTC modulated the expression of some genes in a manner essentially identical to that exhibited by α-tocopherol. These findings support the notion that α-tocopherol modulates gene expression via an antioxidant-independent mechanism.

Keywords: gene expression; lipids; tocopherol; vitamin E; vitamins.

Figure 1

6-HMTC is an analog of vitamin E with no antioxidant activity in vitro.A, chemical structures of α-tocopherol (top) and of 6-HMTC (bottom). B, rationale for the poor radical-trapping activity of 6-HMTC. Shown are possible hydrogen-atom abstractions by lipid peroxyl radicals from benzylic positions of (A) alpha-tocopherol, (B) 6-HMTC, and (C), from the benzylic alcohol of 6-HMTC. A, the established mechanism of tocopherol acting as a chain-terminating antioxidant. B, the possible sites of benzylic H-atom abstraction from 6-HMTC. C, depicts how the alcohol of 6-HMTC has the highest BDE. C, schematic scheme for the synthesis of 6-HMTC. See Supporting Information for details. D, high-affinity binding of 6-HMTC to the α-tocopherol transfer protein (TTP). Purified recombinant TTP (0.4 μM) was preincubated with 1.6 μM NBD-C9-α-Toc in 250 mM sucrose, 100 mM KCl, 50 mM Tris, 1 mM EDTA, pH 7.4. Incremental amounts of unlabeled α-tocopherol (empty circles) or 6-HMTC (solid circles) were added, and fluorescence values recorded for each addition after equilibrium were reached (15 min). BDE, bond dissociation enthalpy; 6-HMTC, 6-hydroxymethyl α-tocopherol.

Figure 2

6-HMTC has no antioxidant activity in vitro and in vivo.A, coautoxidation of egg phosphatidylcholine lipids (1 mM) and STY-BODIPY (8 μM) suspended in phosphate-buffered saline, pH 7.4 was initiated by di-tert-undecylhyponitrite (DTUN; 0.2 mM) at 37 °C. Oxidation was inhibited by the presence of 10 μM α-tocopherol (blue), whereas 6-HMTC (red) did not impact lipid oxidation under the same conditions. B, coautoxidation of styrene (4.3 M) and PBD-BODIPY (10 μM) was initiated by AIBN (6 mM) in phenyl chloride at 37°C and was delayed in the presence of 2 μM α-tocopherol (blue). 6-HMTC (red) was ineffective in delaying lipid oxidation under the same conditions. C, accumulation of lipid hydroxides was induced in cultured IHH cells by treatment with RSL-3, and intracellular reactive oxygen species were measured using the cell-permeable fluorescent probe CM-H2DCFDA (2′,7′-dichlorofluorescin diacetate; 20 μM). Where indicated, cells were preincubated with 50 μM α-tocopherol, 6-HMTC, or control solvent (p < 0.01). D, 6-HMTC does not protect cells from RSL-3-induced ferroptosis. Human embryonic kidney 293 cells were pretreated with the indicated concentrations (0, 0.25, 0.5, 1, 2.5, 5, 10, 20, and 40 μM) of α-tocopherol (solid circles) or 6-HMTC (empty circles) prior to incubation with RSL-3 (0.45 μM; 3.5 h). Viability was measured using the AquaBluer assay (MultiTarget Pharmaceuticals) according to the manufacturer's instructions. Cell viability was calculated by normalizing the data to untreated controls. Each experiment was carried out in six analytical replicates per concentration and repeated independently three times. 6-HMTC, 6-hydroxymethyl α-tocopherol; AIBN, azobisisobutyronitrile; IHH, immortalized human hepatocyte; RSL-3, Ras-selective lethal 3.

Figure 3

Transcription of the TTPA gene is enhanced by α-tocopherol as well as 6-HMTC. Triplicate IHH cultures were treated with the indicated compound for 24 h prior to RT–PCR analyses of the TTPA transcript as described in Experimental procedures section. Transcript abundance was normalized to that of a housekeeping transcript (GAPDH) and then normalized to the value obtained for untreated cells. DFX (deferoxamine) is a hypoxia-inducing iron chelator that was previously shown to increase TTPA levels (62). Shown are means ± standard deviations of the data. 6-HMTC, 6-hydroxymethyl α-tocopherol; IHH, immortalized human hepatocyte.

Figure 4

Differential gene expression profiles of vitamin E- and 6-HMTC-treated IHH cells. IHH cells were treated with 50 μM α-tocopherol, 50 μM 6-HMTC, or dimethyl sulfoxide control for 24 h and subjected to RNA-Seq. A, Venn diagram summarizing transcriptome changes in each treatment group. B, volcano plot illustrating the statistical significance and fold change of each gene in the a-tocopherol versus control contrast. The top 25 upregulated and top 25 downregulated genes that were differentially expressed across both the 6-HMTC versus control and the α-tocopherol versus control groups are labeled. C, protein–protein interaction (PPI) network illustrating the relationship between all 158 statistically significant differentially expressed genes shared amongst the 6-HMTC versus control and a-tocopherol versus control contrasts. K-means clustering was used to define discrete clusters, each colored in different colors. D, heatmap showing the top predicted transcription factors that could mediate the changes in transcript levels observed within each contrast. 6-HMTC, 6-hydroxymethyl α-tocopherol; IHH, immortalized human hepatocyte.