Additional file 3: Figure S3. of The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells

Results of HeLa cell-derivatives EB8 and HeEB1. (A) Oxygen consumption rate of HeLa cells, EB8 and HeEB1cells were demonstrated. (B) Graphic depiction of reactive oxygen species (ROS) production in HeLa cells and EB8 cells exposed to the indicated concentrations of lidocaine (0, 4, or 10 mM) for 6 h (n = 3). Data depict the ratio of ROS production in treated cells compared to that in the untreated control group (HeLa cells). (C) Activities of Caspase3/7 of HeLa cells and HeEB1 cells were demonstrated. (D) Levels of cell death were measured using an Annexin V-FITC Apoptosis Detection Kit evaluated by FACS were demonstrated. (E) Graphic depiction of the levels of cell death among treated and untreated cell populations. Cell death was evaluated by measuring the levels of lactate dehydrogenase (LDH) within culture supernatants (n = 3) in the presence or absence of 10 mM N-acetyl cysteine (NAC), 250 μM Trolox and 10 μM GGA. Control is LDH activity treated by lysis buffer. (F) Graphic depiction of reactive oxygen species (ROS) production in SH-SY5Y cells exposed to 4 mM) for 6 h (n = 3) in the presence or absence of 10 mM N-acetyl cysteine (NAC), 250 μM Trolox and 10 μM GGA. Data depict the ratio of ROS production in treated cells compared to that in the untreated control group. Data presented in A–E expressed as means ± standard deviations (SD). #p < 0.05 compared with the control cell population at the same time period. (PDF 587 kb

Propofol induces a metabolic switch to glycolysis and cell death in a mitochondrial electron transport chain-dependent manner

<div><p>The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia and sedation in critical patient care. However, the rare but severe complication propofol infusion syndrome (PRIS) can occur, especially in patients receiving high doses of propofol for prolonged periods. <i>In vivo</i> and <i>in vitro</i> evidence suggests that the propofol toxicity is related to the impaired mitochondrial function. However, underlying molecular mechanisms remain unknown. Therefore, we investigated effects of propofol on cell metabolism and death using a series of established cell lines of various origins, including neurons, myocytes, and trans-mitochondrial cybrids, with defined mitochondrial DNA deficits. We demonstrated that supraclinical concentrations of propofol in not less than 50 μM disturbed the mitochondrial function and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting mitochondrial complexes I, II and III. This disturbance in mitochondrial electron transport caused the generation of reactive oxygen species, resulting in apoptosis. We also found that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted propofol-induced caspase activation and cell death induced by clinical relevant concentrations of propofol in not more than 25 μM. With further experiments with appropriate <i>in vivo</i> model, it is possible that the processes to constitute the molecular basis of PRIS are identified.</p></div

Propofol induced cell death and decreased mitochondrial membrane potential in a concentration- and time-dependent manner.

<p>(A) SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, 100, or 150 μM) of propofol for 6 h and 12 h. LDH release was assayed in culture supernatants (n = 3). Treatment with lysis buffer served as a control. (B) Average mitochondrial membrane potential (ΔΨm) of untreated SH-SY5Y cells and SH-SY5Y cells treated with the indicated concentrations (25, 50, or 100 μM) of propofol (n = 3) for 6 h. Values indicate the ratio [Q2/(Q2 + Q4)] of green JC-1 monomers (527 nm emission) to red aggregates (590 nm emission). Data are expressed as the mean ± SD. Differences between treatment groups were evaluated by two-way ANOVA, followed by Tukey's multiple comparison test (A), or by one-way ANOVA, followed by Tukey's multiple comparison test (B). *<i>p</i> < 0.05 compared to the control cell population (incubation for 0 h, no treatment).</p

Involvement of functional mitochondria in propofol-induced caspase activation and cell death.

<p>P29 and ρ0P29 cells lacking mtDNA were exposed to the indicated concentrations (25, 50 or 100 μM) of propofol for 6 h. (A) Cells were harvested, and percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>) (n = 3). (B) Caspase-3/7 activity in each treatment group (n = 3) at 6 h. Differences between treatment groups were evaluated by one-way ANOVA, followed by Tukey's multiple comparison test. *<i>p</i> < 0.05 compared to the control cell population; #<i>p</i> < 0.05 compared to the indicated experimental groups.</p

Oxygen metabolism and ROS generation in SH-SY5Y cells and P21 cells treated with propofol.

<p>OCR (A, C, and E) and ECAR (B, D, and F) in SH-SY5Y cells or P29 cells exposed to the indicated concentrations of propofol (12.5, 25, 50, or 100 μM) for 6 h (A, B, E and F, respectively) or 0, 3, 6, or 12 h (C and D). Data presented are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison test (A, B, E and F), or by two-way ANOVA, followed by Dunnett’s multiple comparison test (C and D). (G) ROS production was measured in SH-SY5Y cells exposed to 25, 50, or 100 μM propofol (n = 3) for 3 h or 6 h. (H) SH-SY5Y cells were exposed to the indicated concentrations (50 or 100 μM) of propofol for 6 h with or without treatment with 10 mM <i>N</i>-acetylcysteine. Cells were harvested, and percentages of cell death were measured by flow cytometry. MFI: median fluorescence intensity; NAC: <i>N</i>-acetylcysteine. Data presented are expressed as the mean ± SD. Differences between treatment groups were evaluated by two-way ANOVA, followed by Tukey's multiple comparison test (G), or by one-way ANOVA, followed by Tukey's multiple comparison test (H). *<i>p</i> < 0.05 compared to the control cell population.</p

Synergistic effects of propofol and metformin on caspase activity and cell death.

<p>(A) OCR and (B) ECAR of SH-SY5Y cells exposed to the indicated concentrations of metformin (2.5, 5, 10, or 20 mM) with or without propofol for 6 h. (C) SH-SY5Y cells were exposed to 25 μM propofol with or without 5 mM metformin for 6 h, and ROS production was determined (n = 3). (D and E) SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, or 100 μM) of propofol with or without 5 mM metformin for 6 h. (D) Cells were harvested, and percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (n = 3) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>). (E) Caspase-3/7 activity in each treatment group (n = 3). Data presented in (A–E) are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison test (A, B, and C), or by two-way ANOVA, followed by Tukey's multiple comparison test (D and E). *<i>p</i> < 0.05 compared to the control cell population; #<i>p</i> < 0.05 compared to the indicated experimental groups.</p

Effects of propofol on OCR driven by each complex of the mitochondrial ETC.

<p>Representative OCR traces of mitochondrial respiration using protocol A (Fig A and B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s007" target="_blank">S4 Fig</a>) and protocol B (Fig C and D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s007" target="_blank">S4 Fig</a>). Mitochondrial ETC-mediated OCR, driven by complexes I (A), II (B), III (C), and IV (D), were assayed using an extracellular flux analyzer. SH-SY5Y cells were exposed to 50 or 100 μM propofol for 6 h and subjected to the assay. Differences between treatment groups were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison test. *<i>p</i> < 0.05 compared to the control cell population.</p

Propofol induced cell death and caspase activation in a concentration- and time-dependent manner.

<p>SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, 100, or 150 μM) of propofol for 6 h (A) and 3, 6, and 12 h (B). Cells were harvested, and percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (A and B) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>) (n = 3). SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, 100, or 150 μM) of propofol for 6 h (C and D) and 3, 6, and 12 h (E). Caspase-9 (n = 5) (C) and caspase-3/7 (n = 5) (D and E) activities were assayed in each treatment group at different time points. (F, G and H) C2C12 cells (F), HeLa cells (G) and P29 cells (H) were exposed to the indicated concentrations (12.5, 25, 50, 100 or 150 μM) of propofol for 6 h. The graphical depiction of caspase-3/7 activity is shown (n = 3). Data presented are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Tukey's multiple comparison test (A, C, D, F, G and H), or by two-way ANOVA, followed by Tukey's multiple comparison test (B and E). *<i>p</i> < 0.05 compared to the control cell population (incubation for 0 h, no treatment).</p

Synergistic effects of propofol and mitochondrial ETC inhibitors on caspase activity and cell death.

<p>Levels of caspase-3/7 activity and cell death in SH-SY5Y cells treated with propofol and mitochondrial ETC inhibitors. Cells were treated with 12.5 or 25 μM propofol and with either 100 nM rotenone, 4 μM oligomycin, or 25 μg/mL antimycin A for 6 h and subjected to (A) a cell death assay and (B) a caspase-3/7 activity assay (n = 3). Percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>) (n = 3). All data are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Tukey's multiple comparison test. *<i>p</i> < 0.05 compared with control cells (no treatment); #<i>p</i> < 0.05 compared with the indicated groups. rot: rotenone; olig: oligomycin; anti: antimycin A.</p

Effects of propofol on caspase activity and cell death in various transmitochondrial cybrid cells.

<p>(A) OCR and (B) ECAR of P29, its cybrid cells, and ρ0P29 cells. (C and D) P29, its cybrid cells, and ρ0P29 cells were exposed to the indicated concentrations (12.5, 25, or 50 μM) of propofol for 6 h. Cells were harvested, and percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (n = 3) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>) (C). Caspase-3/7 activity in each treatment group were assayed (n = 3) (D). (E) P29, P29mtA11 and ρ0P29 cells were exposed to 25 or 50 μM propofol for 6 h and subjected to ROS assay. (F) P29mtA11 cells were exposed to 25 μM propofol with or without 10 mM NAC for 6 h. Cells were harvested, and percentages of cell death were measured by flow cytometry. Data presented in (A–F) are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Tukey's multiple comparison test (A and B), by Dunnett’s multiple comparison (E and F) or by two-way ANOVA, followed by Tukey's multiple comparison test (C and D). *<i>p</i> < 0.05 compared to the control cell population; #<i>p</i> < 0.05 compared with the indicated groups. A11: P29mtA11 cells; B82M: P29mtB82M cells; COIM: P29mtCOIM cells; mtΔ: P29mtΔ cells.</p