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. Author manuscript; available in PMC: 2015 Dec 2.
Published in final edited form as: Int J Mol Med. 2011 Aug 5;28(5):711–720. doi: 10.3892/ijmm.2011.765

Cytoprotective effect of γ-tocopherol against tumor necrosis factor-alpha induced cell dysfunction in L929 cells

Gabor Oláh 1, Katalin Módis 1, Domokos Gerő 1, Kunihiro Suzuki 1, Douglas DeWitt 1, Daniel L Traber 1, Csaba Szabó 1
PMCID: PMC4667715  NIHMSID: NIHMS740228  PMID: 21822532

Abstract

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 alpha (TNF-α) 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) + TNF-α (100 pg/ml) in the absence or presence of 1 hour of γ-tocopherol pretreatment. The earliest change that was detected in our system in response to TNF-α was an increase in mitochondrial oxidant production, already apparent at 45 minutes. Changes in glycolysis and oxidative phosphorylation parameters were already apparent at 2h, as detected by the Seahorse Biosciences XF24 Flux Analyzer. By 6 hours, 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 detected by the MTT method, a significant of increase in LDH release into the culture medium, and a detectable degree of mitochondrial membrane depolarization. Between 12 hours and 24 hours, the cell viability (already at a low level) further declined, which coincided with a secondary, marked decline in mitochondrial membrane potential. Pretreatment of the cells with γ-tocopherol (10 μM - 300 μM) provided a significant protection against all of the functional alterations induced by actinomycin D and TNF-α. 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.

Keywords: antioxidants, cell death, necrosis, apoptosis, PARP, reactive oxygen species, vitamin E

Introduction

Multiple studies have demonstrated the potent anti-oxidant effects of γ-tocopherol, the minor component of the antioxidant vitamin E. As opposed to α-tocopherol (which is the principal component of Vitamin E), γ-tocopherol exerts protective effects against both reactive oxygen and reactive nitrogen species in various cell types, resulting in potent cytoprotective effects in various experimental systems (1-3).

The biochemical reactions and the potential therapeutic utility of γ-tocopherol has become a subject of intensive investigations in recent years (4). Several studies have demonstrated the therapeutic potential of γ-tocopherol in various models of critical illness as well as in various chronic inflammatory and metabolic diseases (4-6).

The translational relevance of γ-tocopherol is underlined by recent studies demonstrating the depletion of this antioxidant in severe burn injury and other disease conditions (7-8) association of high levels of this antioxidant with disease prevention (8,9), and therapeutic effects of tocopherol supplementation in pilot clinical studies (10,11).

Incubation of cultured cells with the pro-inflammatory cytokine tumor-necrosis factor alpha (TNF-α) has been used in multiple studies to characterize the mechanisms of inflammatory cell injury. In L929 cells TNF-α is often combined with the RNA synthesis inhibitor actinomycin D, which potentiates the degree of cell death/cell injury (e.g. 12-15). The current investigation had two main goals. The first goal was to explore the contribution of reactive oxygen species (ROS) to the development of cell dysfunction in this commonly used model of cell injury. The second goal of the study was to test the potential cytoprotective effect of the antioxidant γ-tocopherol in this model. The results demonstrate a strong pro-oxidative component of the pro-inflammatory cell death in this model system, and the marked cytoprotective effect of γ-tocopherol.

Materials and Methods

Materials

All reagents (unless specified otherwise) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Cell culture

L-929 mouse fibroblasts cell-line was maintained in 25 mM glucose containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C in a 5% CO2 atmosphere. For the metabolic studies L-929 cells were seeded into special 24-well Seahorse Bioscience V7 tissue culture plates to the indicated density and allowed to adhere and grow for 24 h. Cells were treated with TNF-α (recombinant human TNF-α at a final concentration of 100 pg/ml) and actinomycin D (at a final concentration of 0.5 μg/ml) in the absence or presence of 1 hour of γ-tocopherol (in most experiments, at a final concentration of 100 μM) on 96-well plates. In a series of concentration-response studies, the effect of γ-tocopherol (alone or as a pretreatment before actinomycin D and TNF-α) was also evaluated at concentrations of 1 μM - 1 mM. In a separate set of experiments, the effect of γ-tocopherol (1 μM - 1 mM) was tested on the cytotoxicity elicited by TNF-α alone, when applied alone, at a higher concentration of 1000 pg/ml. Analysis was performed at various times thereafter (45 min - 24 h), depending on the experimental protocol.

Measurement of cell viability using the MTT method

To estimate the number of viable cells 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the cells at a final concentration of 0.5 mg/ml and cultured at 37°C for 1 hour. Cells were washed with PBS and the formazan dye was dissolved in isopropanol. The amount of converted formazan dye was measured at 570 nm with background measurement at 690 nm on a Spectramax microplate reader (Molecular Devices, Mountain View, CA) (16). Viable cell count was calculated as percent of vehicle-treated control cells.

Measurement of cell death using the LDH method

Cell culture supernatant (50 μl) was mixed with 100 ul freshly prepared LDH assay reagent to reach final concentrations of 85 mM lactic acid, 1040 mM nicotinamide adenine dinucleotide (NAD+), 224 mM N-methylphenazonium methyl sulfate, 528 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 200 mM Tris (pH 8.2). The plates were incubated for 15 min and read at 492 nm with background measurement at 690 nm (endpoint assay) on a microplate reader as described (16). LDH activity was expressed as percent of LDH activity measured in the supernatant of vehicle-treated control cells.

Measurement of mitochondrial membrane potential using the JC-1 method

To monitor mitochondrial membrane potential, 5,5′,6,6′-Tetrachloro-1,1′,3,3′- tetraethylbenzimidazolo-carbocyanine iodide (JC-1) was used as described (17). The green fluorescent JC-1 probe exists as a monomer at low membrane potential. However, at higher potentials, JC-1 forms red-fluorescent aggregates. Thus, the emission of this cyanine dye can be used as a sensitive measure of membrane potential. In healthy cells, JC-1 exists as a monomer in the cytosol (green fluorescent signal) and also accumulates as aggregates in the mitochondria (red fluorescence signal). In apoptotic and necrotic cells, more JC-1 exists in monomeric form and stains the cytosol green. The ratio of green-red fluorescence is a parameter that is dependent on the membrane potential. After treatment, cells were first loaded with JC-1 for half an hour at 37°C in DMEM than washed with PBS. Reading was carried out in PBS. Fluorescence intensity measurements were made with BioTek plate reader with the following settings: Ex: 485/530, Em: 528/590.

Measurement of mitochondrial ROS production using MitoSox Red

MitoSOX™ Red mitochondrial superoxide indicator is a fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells. MitoSOX™ Red reagent is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence. MitoSOX™ Red reagent is readily oxidized by superoxide but not by other reactive oxygen or reactive nitrogen species –generating systems, and oxidation of the probe is prevented by superoxide dismutase (18). After treatment cells were washed and loaded with 5 μM of staining solution (in DMEM) for 30 min at 37°C. Cells were washed and 100 μl PBS were added. Fluorescence read was performed on a microplate reader (BioTek), Ex/Em: 510/580.

Cell impedance measurements using the XCelligence method

To monitor cellular events in real time without the incorporation of labels we used the XCelligence system to measure electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates. The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability, morphology and adherence. Cells (20.000 cells/well) were seeded into 180 μl of media in a 96X E-Plate. The attachment, spreading and proliferation of the cells were monitored every 15 minutes using the XCelligence® system as described (16). Approximately 24 hours after seeding, cells were treated with addition of 20 μl test compounds dissolved in cell culture media.

Bioenergetic measurements using the Seahorse method

An XF24 Analyzer (Seahorse Biosciences) is used to measure bioenergetic function in intact L-929 cells. The XF24 creates a transient 7 μl chamber in specialized microplates that allows for OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) to be monitored in real time (19). For all bioenergetic measurements, the culture medium was changed 1 hour prior to the assay run to unbuffered 25 mM glucose containing DMEM (pH 7.4) supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate. First, the optimal number of L-929 cells per well was determined to allow the appropriate detection of changes in OCR and ECAR for the subsequent experiments as 50,000 cells per well. Next, an assay protocol was developed to measure indices of mitochondrial function. TNF-α plus actinomycin D, oligomycin, FCCP and antimycin A were injected sequentially through ports of the Seahorse Flux Pak cartridges to reach final concentrations of 100 pg/ml plus 0.5 μg/ml, 1 μg/ml, 0.3 μM and 2 μg/ml, respectively. 100 μM final concentration of γ-tocopherol was applied as a pretreatment 1 hour prior to the administration of TNF-α and actinomycin D. Oligomycin, FCCP and antimycin A, when used in the appropriate manner, as previously described (20) allow the determination of the basal level of oxygen consumption, the amount of oxygen consumption linked to ATP production, the level of non-ATP-linked oxygen consumption (proton leak) as well as the maximal respiration capacity and the non-mitochondrial oxygen consumption. The values of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) reflect the metabolic activities of the cells and the number of cells.

Immunoblot analysis

Cells treated with TNF-α and actinomycin D in the presence or absence of γ-tocopherol (as above) and processed for Western blotting of poly(ADP-ribose), the product of the nuclear enzyme PAR as described (21). Cells were washed once in PBS and collected by scraping into 100 μl ice-cold RIPA buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, and a cocktail of protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin). Supernatants were collected after centrifugation. Protein was loaded onto 4-12 % polyacrylamide gels. Proteins were separated by electrophoresis and then transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 10% nonfat dried milk in Tris-buffered saline-Tween 20 (TBST) for 90 min. Primary antibodies against PAR (Trevigen, anti-rabbit), and mouse monoclonal alpha-actin (for loading control) were applied at 1 μg/ml concentrations overnight at 4°C. After washing 3 times in TBS containing 0.05% Tween-20 (TBST), secondary antibodies (peroxidase-conjugated goat anti-mouse and goat anti-rabbit) were applied at 1:2000 dilution for 1 h. Blots were washed 3 times in TBST, once in TBS, and incubated in enhanced chemiluminescence reagents (Pierce ECL Western Blotting Substrate) and signals were detected and analyzed using the Syngene Detection system (GBox) with a corresponding software package (GeneSnap).

Statistical Analysis

Summary statistics of data are expressed as mean ± SEM. Comparisons were made using two-way analysis of variance with a Tukey-Kramer post hoc procedure. P-values of ≤ 0.05 (*) and ≤ 0.01 (**) were considered statistical significant.

Results

Time-course of the TNF-α + actinomycin D induced cellular responses

Fig. 1. depicts the changes in viability in response to actinomycin D + TNF-α, using two standard methods, the MTT reduction method and the LDH release method. Actinomycin D + TNF-α induced a deterioration of all three parameters, indicative of cell dysfunction. However, the two methods showed a different time-course of the response. None of the parameters measured have changed significantly for the first 3 hours. At 6 hours, there was a slight trend in decrease in MTT, concomitant with a slight, but statistically significant increase in the LDH content into the supernatant of the cells. By 12 hours, the MTT method showed a marked suppression of mitochondrial respiration (over 80% decrease from control); the LDH content of the supernatant continued to rise (an approximately doubling of the value from the value detected at 6 hours). Between 12 and 24 hours, the already low MTT values showed a slight further decline. The LDH levels in the supernatant did not increase any further over the same time period.

Fig. 1.

Fig. 1

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γ-tocopherol reduces L929 cell injury in response to actinomycin D and TNF-α in L929 cells. Confluent L929 cultures were pretreated with γ-tocopherol (100 μM) or vehicle and subjected to combined actinomycin-D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) for the indicated time periods. Viability was determined by (A) the MTT assay and (B) the LDH assay. There was a progressive deterioration of both viability parameters after AcD/TNF-α, compared to untreated control (CTL) cells (**P < 0.01) and pretreatment with γ-tocopherol significantly protected against these alterations (##P < 0.01). Data are shown as mean ± SEM values of n=9 wells collected from n=3 experiments performed on 3 different experimental days.

Time-course of the mitochondrial membrane depolarization and mitochondrial superoxide production after TNF-α + actinomycin D treatment

Mitochondrial membrane depolarization (as measured by the JC-1 method) did not commence until well after the mitochondrial ROS production has already began: there was no change in JC-1 staining at 3 hours after stimulation, while there was a slight increase at 6-12 hours (Fig. 2). However, there was a significant and marked further increase in mitochondrial membrane depolarization between 12 and 24 hours (Fig. 2). Mitochondrial superoxide production, as measured by the MitoSox Red method, showed an early increase (measured at 45 minutes after stimulation). Among all of the parameters studied in the current series of experiments, this change was the earliest detected alteration. The degree of superoxide production increased by 90 minutes, plateaued by 3 hours (Fig. 3), and failed to further increase throughout the rest of the experimental period (data not shown).

Fig. 2.

Fig. 2

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γ-tocopherol preserves mitochondrial membrane potential (ΔΨm) in response to actinomycin D and TNF-α in L929 cells. Confluent L929 cultures were pretreated with γ-tocopherol (100 μM) or vehicle and subjected to combined actinomycin D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) for the indicated time periods. Mitochondrial membrane potential was evaluated by measuring the green/red fluorescent ratio of JC-1. There was a progressive deterioration of mitochondrial membrane potential after AcD/TNF-α, compared to untreated control (CTL) cells (**P < 0.01) and pretreatment with γ-tocopherol significantly protected against these alterations (##P < 0.01). Data are shown as mean ± SEM values of n=9 wells collected from n=3 experiments performed on 3 different experimental days.

Fig. 3.

Fig. 3

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γ-tocopherol reduces mitochondrial superoxide production in response to actinomycin D and TNF-α in L929 cells. Confluent L929 cultures were pretreated with γ-tocopherol (100 μM) or vehicle and subjected to combined actinomycin-D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) for the indicated time periods. Mitochondrial superoxide production was measured by the MitoSOX Red method. AcD/TNF-α induced a rapid increase in mitochondrial superoxide production, compared to untreated control (CTL) cells (**P < 0.01) and pretreatment with γ-tocopherol significantly reduced this response (##P < 0.01). Data are shown as mean ± SEM values of n=9 wells collected from n=3 experiments performed on 3 different experimental days.

Time-course of the changes in Cell Index, as measured by the XCelligence method after TNF-α + actinomycin D treatment

There were no changes in Cell Index until approximately 6 hours, at which time point this parameter began to decline (Fig 4), indicative of an increased cellular conductance, most likely due to a loss of paracellular connections due to an early cell dysfunction. Cell Index substantially declined by 12 hours, and showed a slight additional decline in the time period thereafter. The time course of these changes most closely paralleled the changes in MTT after actinomycin D + TNF-α in the current experimental system (Fig. 1a vs. Fig. 4). In this experimental system we have also tested a dose-response to actinomycin D and TNF-α, and found that substantial (up to 10-fold) increases in TNF-α concentration only result in a slight enhancement of the response. However, even in response to these elevated concentrations of TNF-α + actinomycin D, the cells did not show any noticable change in Cell Index for until approximately 5-6 hours after stimulation. A decrease in TNF-α and actinomycin D concentration to 30 or 10 pg/ml, on the other hand, resulted in a markedly diminished cytotoxic response (Fig. 4).

Fig. 4.

Fig. 4

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γ-tocopherol reduces the changes in Cell Index in response to actinomycin D and TNF-α in L929 cells. Confluent L929 cultures were pretreated with γ-tocopherol (100 μM) or vehicle and subjected to combined actinomycin-D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) for the indicated time periods. Cell Index, as a biophysical indicator of cell impedance, was measured by the XCelligence method. AcD/TNF-α induced a decline in Cell Index, compared to untreated control (CTL) cells and pretreatment with γ-tocopherol prevented this response. A representative experiment representing mean values of n=3 wells per condition is shown for each experimental groups. Similar results were found in 3 independent experiments performed on different experimental days.

Bioenergetic changes after actinomycin D + TNF-α administration

After obtaining the basal respiration rate in each group, cells were treated with actinomycin D and TNF-α and mitochondrial and glycolytic activities were monitored for 10 hours. Actinomycin D and TNF-α induced an immediate decline in the oxygen consumption rate (OCR) and the cellular glycolytic rate (ECAR) that stabilized at 2 hours after the administration of actinomycin D + TNF-α (Fig. 5a). While these drops in the OCR and ECAR were relatively modest, actinomycin D + TNF-α significantly diminished the mitochondrial respiratory reserve capacity and glycolytic rate of the cells as assessed by the mitochondrial uncoupling agent FCCP at 6 hours (Fig. 5b A-D). It is important to point out that all of these energetic alterations occurred in a time period at which the more conventional viability assays (MTT, LDH) or the XCelligence-based detection of cell conductance have not yet demonstrated any overt alterations, and at which time point no marked mitochondrial membrane depolarization (as detected by JC-1) has yet occurred. These observations are consistent with the hypothesis that an early oxidative stress response, induced by actinomycin D + TNF-α, leads to a substantial, simultaneous defect of both oxidative phosphorylation and glycolysis in L929 cells, which already begins at an early time at which no overt cell toxicity or cell dysfunction is apparent.

Fig. 5.

Fig. 5

Fig. 5

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Measurement of mitochondrial function in L-929 cell cultures using the Seahorse XF24 analyzer; effect of γ-tocopherol on the bioenergetic changes induced by actinomycin D and TNF-α. Time course for measurement of OCR and ECAR for 50,000 cells under the basal condition followed by the sequential addition of actinomycin D + TNF-α (AcD/TNF-α), oligomycin (1 μg/ml), FCCP (0.3 μM), and antimycin A (2 μg/ml). Part A of Figure 5a represents OCR values and part B represents ECAR values, both expressed as percentage of baseline. In Figure 5b AUC analyses are shown. These were used to determine the overall amount of oxygen consumption (AUC OCR %) during the treatment of FCCP (A). Based on the AUC OCR % data the calculated mitochondrial reserve capacity compared to CTL group is also shown (B). Furthermore, the AUC analyses were also applied to determine the overall amount of acidification rate (AUC ECAR %) or proton production after the administration of FCCP (C). Based on the AUC ECAR % data the calculated glycolytic rate is shown in part (D). AcD/TNF-α induced a decrease in both OCR and ECAR parameters (*p<0.05, **p<0.01) and pretreatment with γ-tocopherol attenuated these alterations (#p< 0.05, ##p< 0.01). Data are shown as mean ± SEM values of n = 15 wells collected from n = 5 experiments performed on 3 different experimental days.

The ATP production coupled oxygen consumption was also determined with the administration of ATP synthase inhibitor oligomycin using the Seahorse XF24 Analyzer. The oxygen consumption rate coupled to ATP production was severely diminished in response to actinomycin D + TNF-α. (Fig. 5b E-F). Actinomycin D + TNF-α reduced the OCR linked to ATP production by 50% compared to the controls. Actinomycin D and TNF-α reduced both the OCR and the ECAR by 40%, while γ-tocopherol exerted a significant protection against these alterations. Tocopherol restored the mitochondrial reserve capacity to approximately 90% of controls, whereas proved less efficient to revert the ECAR values representing the glycolytic changes in the cells. Taken together, the above observations are consistent with the hypothesis that an early oxidative stress response, induced by actinomycin D + TNF-α, leads to a substantial, simultaneous defect of both oxidative phosphorylation and glycolysis in L929 cells, which already begins at an early time at which no overt cell toxicity or cell dysfunction is apparent.

PARP activation after actinomycin D + TNF-α administration

Activation of PARP, a nuclear enzyme, has been implicated in many forms of oxidative cell injury (Jagtap and Szabó, 2005), as well as in the current model of actinomycin D + TNF-α induced cell dysfunction/cell death in L929 cells (13). Consistent with these observations, we have detected a substantial increase in PARylation of the cells. The most pronounced increase was seen at 24 hours, which was a doubling of the PARylation response (Fig. 6); at 12 hours the degree of the increase in PARylation was approximately 50% of the value seen at 24 hours, whereas at 1, 3 and 6 hours no significant increase in PARylation was detected (data not shown).

Fig. 6.

Fig. 6

Fig. 6

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γ-tocopherol reduces PARP activation in response to actinomycin D and TNF-α in L929 cells. Confluent L929 cultures were pretreated with γ-tocopherol (100 μM) or vehicle and subjected to combined actinomycin-D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) for 24 hours. PARP activation was detected by western blotting for poly(ADP-ribose) (PAR), the product of the PARP enzyme. PARP activity is expressed as PAR/actin ratio. Data in part A are shown as mean ± SEM values of n=9 wells collected from n=3 experiments performed on 3 different experimental days. An individual representative Western blot is shown in part B.

Effect of γ-tocopherol on the cellular responses induced by actinomycin D + TNF-α

The presence of γ-tocopherol (100 μM), on its own, failed to influence any of the baseline parameters studied in the current series of experiments. This finding is consistent with the view that the antioxidant, at this concentration (100 μM), is devoid of any intrinsic cytotoxic effects. Pretreatment with γ-tocopherol exerted a significant, and in most cases, complete or near-complete protection against the cellular responses induced by actinomycin D + TNF-α: it completely prevented the suppression of MTT (Fig. 1a), the release of LDH (Fig. 1b), the increase mitochondrial superoxide formation (Fig. 2), the development of mitochondrial depolarization (Fig. 3), the deterioration of Cell Index (Fig. 4), as well as the PARP activation (Fig. 6). As far as the changes in cellular energetics are concerned, γ-tocopherol markedly improved all parameters of oxidative phosphorylation and glycolysis; γ-tocopherol but provided an approximately 75% preservation of the actinomycin D + TNF-α induced alterations in mitochondrial respiratory and glycolytic capacity (Fig. 5). The effect of γ-tocopherol was also tested in a concentration-response experiment on mitochondrial respiration and LDH release (Fig. 7). The optimal cytoprotective concentrations were 100 and 300 μM; at 30 and 10 μM the protective effect diminished, while at the top concentration tested (1 mM), the compound exerted a slight intrinsic suppressive effect on cell viability. In viability assays when cell injury was induced by TNF-α alone (at a higher concentration of 1000 pg/ml), γ-tocopherol continued to exert statistically protective effects starting at the concentration of 10 μM, although, in this series of studies, the extent of the protection remained partial in the entire concentration range of 10-1000 μM tested (Fig. 7).

Fig. 7.

Fig. 7

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γ-tocopherol reduces L929 cell injury in response to actinomycin D and TNF-α in L929 cells: a concentration-response study. Confluent L929 cultures were pretreated with γ-tocopherol (1 μM - 1 mM) or vehicle and subjected to either vehicle treatment (A, B), or combined actinomycin-D (0.5 μg/ml) and TNF-α (100 pg/ml) (AcD/TNF-α) (C, D) or TNF-α alone (1000 pg/ml) (E, F) for 24 h. Viability was determined by the MTT assay (left panels) and the LDH assay (right panel). There was a deterioration of both viability parameters after AcD/TNF-α, or TNF-α compared to untreated control (CTL) cells (**P < 0.01) and pretreatment with γ-tocopherol significantly protected against these alterations (##P < 0.01). γ-tocopherol (at 1 mM, but not at the concentrations tested below) exerted intrinsic cytotoxic effects. Data are shown as mean ± SEM values of n=9 wells collected from n=3 experiments performed on 3 different experimental days.

Discussion

The model of actinomycin D and TNF-α exposure in L929 cells has been often used to study cell death/cell injury in response to pro-inflammatory stimulation (e.g. 12-15). The time-course of the response and many of the cellular effectors have been characterized by multiple experiments. Although the literature is somewhat controversial, according to the more recent body of published studies, the mode of cell death shows many features of necrotic phenotype (22), while caspase activation and apoptotic processes appear to play a minor role (23). The current findings (such as the LDH release into the medium after AcD/TNF-α exposure) are consistent with cell membrane disruption and cell death via the necrotic route.

Mitochondrial generation of reactive oxygen species (24,25) as well as mitochondrial permeability transition (23) have been previously implicated in the pathogenesis of cell injury in the current model. The current findings (early reactive oxygen production, mitochondrial permeability transition) are consistent with these findings. However, it is important to point out that some of the earliest alterations we have observed in response to AcD/TNF-α have occurred at the level of cellular metabolism: both oxidative phosphorylation and glycolysis were found to be impaired in a simultaneous manner, and these alterations appeared to be dependent on the formation of reactive oxidant species (as evidenced by the protection by γ-tocopherol against these alterations). The cellular energetic alterations induced by actinomycin D and TNF-α observed in the current study are consistent with several studies in the literature demonstrating that the oxidant radicals can cause impaired mitochondrial function eventuating in a decrease in respiratory reserve capacity (20,26). Furthermore, many research groups have demonstrated that deleterious oxygen species have crucial role in diminishing mitochondrial function via oxidative impairment of mitochondria, especially complex I (27,28) and complex III proteins (29). It is generally accepted that during oxidative and nitrosative stress, mitochondria are capable of using ‘reserve capacity’ which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. Consequently, impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death.

According to our results, γ-tocopherol pretreatment significantly ameliorated the changes in the respiratory reserve capacity in L929 cultures subjected to actinomycin D and TNF-α, which suggests that γ-tocopherol acts on an upstream process, possibly, at least in part within the mitochondria. The fact that γ-tocopherol is a potent reactive oxygen species scavenger (antioxidant agent) (see: Introduction) which has the potential to reduce oxidative damage in various fields of cellular injury and recent studies demonstrating that γ-tocopherol improves mitochondrial function via reduction of reactive oxygen species produced by mitochondria (30) are consistent with this hypothesis. Indeed, mitochondrial oxidant production was diminished in γ-tocopherol-treated cells exposed to actinomycin D and TNF-α, which may reflect a very effective intramitochondrial scavenger function, a reduced intramitochondrial ROS production or both. In this context it is important to emphasize that we have also demonstrated here that the mitochondrial oxygen consumption is more efficiently coupled to ATP production in the presence of γ-tocopherol that may represent a key step in the mitochondrial reserve capacity preservation and cell survival. We do not have any evidence for, and we do not propose that g-tocopherol selectively concentrate to the mitochondria. Subcellular distribution studies demonstrate that the cellular distribution of tocopherols directly correspond to the lipid distribution (31). Therefore, it is conceivable that some of the g-tocopherol that we applied to the cells will be present in the mitochondrial membrane, and may be localized in the vicinity of the sites where ROS are produced (e.g. the mitochondrial respiratory chain).

In addition to resulting in a decreased level in mitochondrial respiratory capacity, the results obtained with the Seahorse XF24 analyzer also demonstrated that actinomycin D and TNF-α lowers the glycolytic function of the cells. There are several series of data in the literature implicating that reactive oxygen species can diminish GAPDH enzyme function by activating poly(ADP-ribose) polymerase causing reduced GAPDH enzyme activity and subsequently attenuated glycolytic function (32). GAPDH may also be inactivated by S-thiolation induced by respiratory burst or exposure to oxidants (33). According to our data γ-tocopherol exerted a statistically significant protective effect against the actinomycin D and TNF-α induced glycolytic impairment, which is potentially the result of a direct antioxidant effect of the molecule.

Activation of PARP has been shown to contribute to many forms of oxidant-induced cell injury, at least in part via modulation of cellular metabolism, through the activation of suicidal ATP-consuming energetic cycles triggered by oxidative and nitrosative DNA injury (34). In the current model, Agarwal and colleagues have demonstrated an increase in PARP activation and increased poly(ADP-ribosyl)ation (13). However, the time-course of PARP activation (which is not apparent in the first 6 hours) is such, that it is unlikely that it plays a significant role in the early metabolic alterations and in the early phase of cell injury. Based on the results of the previous studies (demonstrating that cellular NAD+ depletion and ATP depletion only starts around 6 hours post actinomycin D and TNF-α exposure, we believe that PARP activation is likely to play a more significant role in the second phase of the cell injury (at 12 - 24 hours). The inhibitory effect of γ-tocopherol on PARP activation is consistent with the hypothesis that the antioxidant vitamin inhibits oxidant-induced DNA injury, which is an obligatory trigger for an increase in the catalytic activity of the constitutively expressed PARP-1 enzyme (the major PARP isoform). These findings are also consistent with in vivo studies in an acute lung injury model showing that treatment of the animals with γ-tocopherol inhibits tissue PARP activation (6). PARP is certainly not the only oxidant-induced downstream pathway of inflammation/cell injury that is triggered by actinomycin D and TNF-α exposure in the current model; several studies demonstrated protein kinase C activation and p38 activation (35). In this context it is noteworthy that in the current model, γ-tocopherol treatment (100 μM) also blocked the phosphorylation of p38, as measured at 24 hours (unpublished data).

Several limitations to the current study need to be acknowledged. First of all, the current study is an entirely in vitro investigation, and, as such, can only mimic to a limited extent the various pathophysiological conditions where γ-tocopherol has been shown to be effective. Therefore, the conclusions made by the current set of studies may not be fully applicable for the in vivo conditions. Second, the current experimental model (combination of a pro-inflammatory cytokine and a transcription inhibitor), even though a frequently used and studied experimental model, does have certain limitations, and does not fully mimic pathophysiological states (where transcriptional inhibition does not normally occur). In order to overcome this limitation, we have also tested the effect of γ-tocopherol in L929 cells exposed to TNF-α alone (in the absence of a transcriptional inhibitor). Although the protection by γ-tocopherol against cell injury in this model was still statistically significant, it was less pronounced than when the protocol utilized the combined application of actinomycin D and TNF-α (Fig. 7). There are substantial differences in the molecular mechanisms of the cell death/cell injury, depending on the absence or presence of protein synthesis inhibitors (15), and we hypothesize that under the conditions of TNF-a alone (where the cytokine needed to be applied at a substantially higher concentration, and even at this concentration, the degree of cell death was less pronounced than when actinomycin D and TNF-α were used together), multiple additional pathways of cell injury and cell death become activated. Some of these pathways (caspases, PARP and other effectors) may not be dependent on oxyradicals, and this may explain the reduced effectiveness of γ-tocopherol. Nevertheless, we must point out that even in this ‘native’ system, γ-tocopherol, at a physiologically and therapeutically relevant concentration (as low as 10 μM) afforded statistically significant cytoprotection, supporting the overall conclusions of the current study.

In summary, the current studies have demonstrated that γ-tocopherol exerts protective effects in cells exposed to various types of oxidative stress, as well as in various in vivo models of acute and chronic diseases which are, at least in part, developing on the basis of an increased oxidative or nitrosative stress (see: Introduction). The current results further underline the antioxidant potency of γ-tocopherol, and may explain some of the protective mode of action for this compound.

Acknowledgments

This work was supported by grants from the National Institutes of Health P01GM06612, 5R01NS19355-17 and R01GM060915 and R01GM056687-11S2.

Abbreviations used

AUC

area under the curve

DTT

dithiothreitol

ECAR

extracellular acidification rate

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimidazolo-carbocyanine iodide

LDH

lactate dehydrogenase

MTT

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

OCR

oxygen consumption rate

PAR

poly (ADP-ribose) polymer

PARP

poly(ADP ribose) polymerase

TNF-α

tumor necrosis factor alpha

Footnotes

Conflict of Interest: Authors declare no conflict of interest.

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