Abstract
BackgroundStimulation of C3H 10T1/2 murine fibroblasts with interferon-γ(IFN) and bacterial lipopolysaccharide (LPS) generates reactive oxygen and nitrogen species leading to DNA damage, lipid oxidation, and tocopherol oxidation. The tocopherols possess unique chemical and biological properties that suggest they have important roles related to intracellular defense against radical-mediated damage.ResultsDespite increased levels of reactive oxidants and decreased media tocopherol, cellular levels of γ-tocopherol, but not α-tocopherol, were observed to increase significantly when cells were treated with IFN/LPS. Inhibition of nitric oxide (NO) synthesis by a specific inhibitor of inducible NO synthase (iNOS) increased both intracellular α-tocopherol and γ-tocopherol concentrations, but did not significantly alter the reduction in media tocopherol levels caused by IFN/LPS treatment. Both exposure to exogenous NO and cellular synthesis of NO in cell culture increased media levels of 8-epi-prostaglandin F2α, a marker of oxidative lipid damage, whereas inhibition of endogenous NO synthesis reduced media 8-epi-prostaglandin F2α formation to control levels.ConclusionElevated intracellular levels of γ-tocopherol in response to the cellular inflammatory state may indicate that it serves a unique role in minimizing cellular damage resulting from endogenous NO synthesis. Results of the current study suggest that NO is an important mediator of damage within the cell, as well as in the oxidation of both α- and γ-tocopherols. The paradoxical increase in cellular tocopherol associated with the induction of NO synthesis may indicate either enhanced cellular transport/decreased export for tocopherols or recruitment of free tocopherol from tocopherol storage molecules.
Highlights
Stimulation of C3H 10T1/2 murine fibroblasts with interferon-γ(IFN) and bacterial lipopolysaccharide (LPS) generates reactive oxygen and nitrogen species leading to DNA damage, lipid oxidation, and tocopherol oxidation
We have shown previously that γ-tocopherol, but not α-tocopherol, enhances cytokine-mediated nitric oxide (NO) synthesis in C3H 10T1/2 cells [43] and that γ-tocopherol more effectively protects against cell death in rat insulinoma cells stimulated to produce NO by interleukin 1-β [12]
Modulation of media and cellular tocopherol levels Preliminary experiments designed to study simultaneous uptake of alpha and gamma tocopherol analogues (5.0 μM each) indicated that tocopherol levels in C3H 10T1/2 cells were stable after seven days with re-treatment (Figure 1) and that no detectable levels of tocopherol were present in the culture medium in the absence of exogenous treatment
Summary
Stimulation of C3H 10T1/2 murine fibroblasts with interferon-γ(IFN) and bacterial lipopolysaccharide (LPS) generates reactive oxygen and nitrogen species leading to DNA damage, lipid oxidation, and tocopherol oxidation. Roles for the tocopherols in human and animal health, beyond those traditionally associated with vitamin E bioactivity, have been identified from epidemiological, clinical and basic laboratory research, suggesting that tocopherols may modulate the development and progression of cardiovascular disease [3,4], cancer [5,6] and neurological abnormalities [2,7], as well as affect immune function [8], natriuresis [9], and inflammation [10] These newly identified functions of the tocopherols appear un-related to their vitamin E associated bioactivity, rather, it is observed that subtle chemical differences in structure and/or function between the various tocopherols are responsible for the different biological properties they manifest [11,12,13]. The absence of definitive evidence from human clinical trials that higher doses of tocopherols have beneficial long-term effects has precluded dietary recommendations for either higher amounts of αtocopherol in the diet or the intake of other tocopherol analogues [2]
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