The antioxidant vitamin γ-tocopherol exerts protective and anti-inflammatory effects in various models of critical illness. The combination of actinomycin D and tumor necrosis factor α (TNFα) in the immortalized fibroblast cell line L929 is a well-established method to model pro-inflammatory cytotoxicity in cultured cells in vitro. The present study had two aims. First, we wished to characterize the contribution of reactive oxygen species (ROS) to the cell dysfunction and this commonly used model system of cell death. Second, we wished to investigate the effects of γ-tocopherol on this response. Cells were exposed to actinomycin D (0.5 μg/ml) + TNFα (100 pg/ml) in the absence or presence of 1 h of γ-tocopherol pre-treatment. The earliest change that was detected in our system in response to TNFα was an increase in mitochondrial oxidant production, already apparent at 45 min. Changes in glycolysis and oxidative phosphorylation parameters were already apparent at 2 h, as detected by the Seahorse Biosciences XF24 Flux Analyzer. By 6 h, a slight decrease in Cell Index was detected by impedance-based analysis, employing an electronic sensor array system (XCelligence). At the same time, a slight decrease in cell viability was detected by the 3-(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method, along with a significant increase in lactate dehydrogenase (LDH) release into the culture medium, and a detectable degree of mitochondrial membrane depolarization. Between 12 and 24 h, the cell viability (already at a low level) furtherdeclined, which coincided with a secondary, marked decline in the mitochondrial membrane potential. Pre-treatment of the cells with γ-tocopherol (10-300 μM) provided a significant protection against all of the functional alterations induced by actinomycin D and TNFα. The current study provides direct evidence that reactive oxidant formation plays an important role in the current experimental model of cell dysfunction, and demonstrates the protective effects of the potent endogenous antioxidant vitamin, γ-tocopherol. The mechanisms described in the current study may, in part, contribute to the protective effects of γ-tocopherol in various models of critical illness.
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