1

The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive2

oxygen species production and cell death in neuronal SH-SY5Y cells

3

4

Akihisa Okamoto1, Hiromasa Tanaka1, Chisato Sumi1, Kanako Oku1, Munenori

5

Kusunoki1, Kenichiro Nishi1, Yoshiyuki Matsuo1, Keizo Takenaga2, Koh Shingu1, and6

Kiichi Hirota1,*

71

Department of Anesthesiology, Kansai Medical University, Hirakata, Japan,

82Department of Life Science, Shimane University School of Medicine, Izumo,

9

Japan

10

Akihisa Okamoto: aokamoto1978@gmail.com ; Hiromasa Tanaka:

1110212291hiro@gmail.com; Chisato Sumi: chisato13150labo@gmail.com ;12

Kanako Oku: sakamaruko0727@gmail.com ; Munenori Kusunoki:13

moo1029taka@gmail.com ; Kenichiro Nishi: nishik@hirakata.kmu.ac.jp ;14

Yoshiyuki Matsuo: matsuo.oustam@gmail.com ; Keizo Takenaga

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biokeizo@med.shimane-u.ac.jp; Koh Shingu k.shingu@jusendo.or.jp ; Kiichi

2

Hirota: hif1@mac.com

3
4*Corresponding Author: Kiichi Hirota Email: hif1@mac.com

5

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Abstract

2

Background: The local anesthetic lidocaine can affect intra- and extra-cellular3signaling pathways in both neuronal and non-neuronal cells, resulting in long-term

4

modulation of biological functions, including cell growth and death. Indeed, lidocaine

5

was shown to induce necrosis and apoptosis in vitro. While several studies have6

suggested that lidocaine-induced apoptosis is mitochondrial pathway-dependent, it

7

remains unclear whether reactive oxygen species (ROS) are involved in this process and8whether the observed cell death can be prevented by antioxidant treatment.

9

Methods: The effects of lidocaine and antioxidants on cell viability and death were10

evaluated using SH-SY5Y cells, HeLa cells, and HeLa cell derivatives. Cell viability

11

was examined via MTS/PES

12([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra

13

zolium, inner salt]/phenazine ethosulfate) assay analysis. Meanwhile, cell apoptosis and14

necrosis were evaluated using an Annexin V-FITC Apoptosis Detection Kit, as well as

15

by assaying for caspase-3/7 and caspase-9 activity, and by measuring the release of

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lactate dehydrogenase, respectively. Mitochondrial transmembrane potential (ΔΨm)

2

was assessed using the fluorescent probe tetramethylrhodamine ethyl ester.

3

Results: Lidocaine treatment resulted in suppression of the mitochondrial electron

4transport chain and subsequent attenuation of mitochondrial membrane potential, as

5well as enhanced ROS production, activation of caspase-3/7 and caspase-9, and6

induction of apoptosis and necrosis in SH-SY5Y cells in a dose- and time-dependent

7

manner. Likewise, the anesthetics mepivacaine and bupivacaine also induced apoptosis

8in SH-SY5Y cells. Notably, the antioxidants N-acetyl cysteine (NAC) and Trolox

9

successfully scavenged the mitochondria-derived ROS and suppressed local

10

lidocaine-induced cell death.

11

Conclusions: Our findings demonstrate that the local anesthetics lidocaine,

12

mepivacaine, and bupivacaine inhibited the activity of mitochondria and induced

13

apoptosis and necrosis in a dose-dependent manner. Furthermore, they demonstrate that

14

treatment with the antioxidants NAC, Trolox, and GGA resulted in preservation of15

mitochondrial voltage and inhibition of apoptosis via suppression of caspase activation.

16

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Keywords: lidocaine, mitochondria, ROS, redox, apoptosis, necrosis, oxygen2

consumption

3
4
5

Background

6

There is an established consensus that local anesthetics exert nerve-blocking activity,

7

primarily through the inhibition of voltage-gated sodium channels [1]. However, these8

compounds can also affect the intra- and extra-cellular signaling pathways of both9neuronal and non-neuronal cells, resulting in long-term modulation of biological10

functions, including cell growth and death [2]. Lidocaine is a widely used local

11

anesthetic and anti-arrhythmic agent. Notably, lidocaine was also shown to induce

12

apoptosis and necrosis both in vitro [3-5] and in vivo [6], and to cause transient or13permanent nerve injury, such as cauda equina syndrome, after spinal anesthesia in

14

clinical settings [7, 8]. In addition, it was reported that lidocaine inhibits the invasive

15

ability of cancer cells at concentrations used for surgical operations (5–20 mM).

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Apoptosis is a form of programmed cell death that is characterized by a series of distinct

2morphological and biochemical changes, and is an important process in a wide variety

3of biological systems. There are two major signaling pathways by which apoptosis is4

induced: the intrinsic and extrinsic pathway. While the extrinsic pathway is dependent

5

on cell-surface death receptors such as Fas (First apoptosis signal), the intrinsic pathway

6

is initiated within mitochondria [9]. Specifically, in the intrinsic pathway, the formation

7of a multimeric Apaf-1/cytochrome c complex results in activation of caspase-9, which

8

in turn cleaves and activates the downstream caspases caspase-3, -6, and -7 [1, 10].

9

Notably, reactive oxygen species (ROS) are widely believed to play an essential role in10

apoptosis. Indeed, several studies indicate that ROS scavengers, including the synthetic

11

compound N-acetyl cysteine (NAC) and the endogenous redox-active molecule12

thioredoxin (TRX), can be used to alleviate intracellular ROS and thereby prevent

13

apoptosis and necrosis.

14

Previous studies demonstrated that lidocaine-induced cell death is dependent on the15

mitochondrial pathway; however, it is still largely unclear whether ROS are involved in

16

this process. In this study, we demonstrate that in vitro lidocaine treatment resulted in

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attenuation of mitochondrial membrane potential and promoted caspase-dependent2

apoptosis in neuronal SH-SY5Y cells. Moreover, we show that the observed increases

3

in cell death were mitochondria-derived ROS-dependent and could be blocked by

4treatment with several antioxidant compounds.

5
6
7

Methods

8

Reagents

9

Lidocaine, mepivacaine, bupivacaine, NAC, and10(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were obtained

11

from Sigma-Aldrich (St. Louis, MO, USA), teprenone (geranylgeranylacetone, GGA)

12

was obtained from Wako Pure Chemical Industries (Osaka, Japan), and recombinant

13

human TRX (rhTRX) was obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan). GGA

14

and Trolox were dissolved in absolute ethanol, while bupivacaine and NAC were15

dissolved in H2O, and rhTRX was dissolved in citric acid. Rotenone, oligomycin and16

antimycin A are obtained from Abcam, Inc. (Cambridge, MA, USA).

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2

Cell culture3

All cell lines were obtained from American Type Culture Collection (ATCC; Manassas,

4

VA, USA). The established cell lines derived from human neuroblastoma SH-SY5Y

5cells and cervical carcinoma HeLa cells were maintained in Roswell Park Memorial

6

Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 100

7units/ml penicillin, and 0.1 mg/ml streptomycin. The characteristics of EB8 cells (HeLa8cells lacking mtDNA) and HeEB1 cells (a hybrid clone of EB8 cells containing mtDNA

9from wild-type HeLa cells) have been described elsewhere [11, 12]. All cells were10

maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were grown11

in 100 mm dishes and were subcultured for experiments when they reached 85%

12confluence.

13
14

Cell viability assay (MTS assay)

15

Cell viability was assessed using a CellTiter 96™ AQueous One Solution Cell

16

Proliferation Assay (Promega, Madison, WI, USA). Briefly, SH-SY5Y cells were

-8-

1

seeded into 96-well plates (2 × 104 cells/well) and cultivated overnight. The following

2

day, cells were treated with the indicated concentrations of the appropriate drug(s) for3

varying lengths of time. After treatment, 20 µl of CellTiter 96 AQueous One Solution™

4

Reagent was added to each well, the plates were incubated at 37°C for 1 h, and the5absorbance of each sample was measured using an iMark™ Micropate Reader

6(BIO-RAD, Hercules, CA, USA) at a wavelength of 490 nm. Cell viability was then7

calculated by comparing the absorbance of treated cells with that of the control cells

8(incubated without drugs), which was defined as 100% [13, 14]. All samples were9

tested in triplicate for each experiment.

10
11

Caspase-3/7 and caspase-9 activity assays

12

The levels of caspase-3/7 and caspase-9 activity were assessed using an Apo-ONE™

13

Homogeneous Caspase-3/7 Assay Kit (Promega) and a Caspase-Glo™ 9 Assays Kit

14(Promega), respectively, according to the manufacturer’s protocols. Briefly, SH-SY5Y

15

cells were seeded into 96-well plates (2 × 104 cells/well) and incubated overnight. The

16

following day, cells were treated with the indicated concentrations of the appropriate

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drug(s) for varying lengths of time. After treatment, 100 µl of Apo-ONE Caspase-3/7

2

Reagent™ or Caspase-Glo 9 reagent™ was added to each well, respectively. Cells were3

incubated at room temperature for 1 h and the luminescence of each well was measured

4using an EnSpire™ Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

5

Caspase activity was then calculated by comparing the levels of luminescence of the6treated cells with that of the control cell population (incubated without drugs), which7

was defined as 100%. Assays were performed in triplicate at least twice. Data were

8

expressed as means ± standard deviations (SD).

910Immunoblot Assays

11

Whole cell lysates were prepared as described previously [15, 16]. Briefly, cells

12were lysed by suspension in ice-cold lysis buffer [0.1% sodium dodecyl sulfate

13(SDS), 1% NP40, 5 mM ethylene diamine tetraacetic acid (EDTA), 150 mM

14

NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, and Complete

15

Protease Inhibitor™ (Roche Applied Science)] and centrifuged at 10,000 × g to16pellet cell debris. Approximately 25 µg of each protein sample was then separated

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by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to2

immunoblot analysis using rabbit polyclonal antibodies specific to PARP [poly3(ADP-ribose) polymerase; 1:1,000] or cleaved caspase-9 (Asp315; 1:1,000) (Cell

4Signaling Technology, Danvers, MA, USA) [17], β-actin (Sigma-Aldrich), and5anti-rabbit IgG horseradish peroxidase-linked secondary antibodies (1:2000

6

dilution; Cell Signaling Technology). Immunolabeled proteins were then7visualized using enhanced chemiluminescence (ECL™) reagents (Amersham

8

Biosciences, Little Chalfont, UK).

9
10

Analysis of cell apoptosis

11

Levels of cell apoptosis were measured using an Annexin V-FITC Apoptosis Detection

12

Kit (BioVision, Milpitas, CA, USA), according to the manufacturer’s instructions. For

13these analyses, SH-SY5Y cells were seeded into 6-well plates (3 × 105 cells/well) and14

incubated overnight. The following day, cells were treated with the indicated15

concentrations of the appropriate drug(s) for varying lengths of time and harvested by

16

centrifugation at 1,200 rpm for 3 min. The culture supernatants were discharged, and

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the resulting pellets were suspended in a mixture comprised of 500 µl binding buffer, 52µl Annexing V-FITC, and 5 µl propidium iodide (PI; 50 µg/ml) for 5 min at room

3

temperature in the dark and analyzed using a FACSCalibur flow cytometer (BD4

Biosciences, San Jose, CA, USA) equipped with CellQuest Pro™ software[4, 13]. Data

5

were evaluated using FlowJo™ version 7.6.3 software (TreeStar, Ashland, OR, USA),

6

exported to Excel spreadsheets, and subsequently analyzed using the statistical

7

application R.

8
9

Lactate dehydrogenase (LDH)-based cytotoxic assay

10

Levels of cell cytotoxicity were evaluated using a CytoTox-ONE™ Kit (Promega).

11

Briefly, SH-SY5Y cells were seeded into 96-well plates (2 × 104 cells/well) and12

incubated overnight. The following day, cells were treated with the indicated13

concentrations of the appropriate drug(s) for varying lengths of time. Twenty

14

microliters of CytoTox-ONE™ reagent was added to each well, plates were incubated15

at 22°C for 10 min, and then 50 µl of Stop Solution was added to each well. The

16

resulting fluorescence was measured using an EnSpire™ Multimode Plate Reader

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(PerkinElmer) at an excitation wavelength of 560 nm and an emission wavelength of2

590 nm. Percentages of cell death were calculated by comparing the level of LDH

3

released (fluorescence value) from each treatment group with that of the positive control

4

population (cells treated with Lysis solution), which was defined as 100%. Meanwhile,5

the level of LDH released from the negative control population (untreated cells) was

6

defined as 0%. All samples were evaluated in triplicate for each experiment.

7
8

Determination of mitochondrial membrane potential (ΔΨm)

9

Mitochondrial membrane potential was determined by flow cytometry using a

10

MitoPT™ JC-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA),11

according to the manufacturer’s instructions. For these analyses, SH-SY5Y cells were12

seeded into 6-well plates (3 × 105 cells/well) and cultivated overnight. The following

13

day, cells were treated with the indicated concentrations of the appropriate drug(s) for

14

varying lengths of time and then pelleted by centrifugation at 1,200 rpm for 3 min.

15

Supernatants were discharged, and cells were resuspended in JC-1, incubated at 37°C

16

for 15 min in the dark, and collected by centrifugation at 1,200 rpm for 3 min.

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Supernatants were again discharged and the remaining cell residues were suspended in

2

500 µl assay buffer. Samples were subsequently analyzed using a FACSCalibur flow3

cytometer (BD Biosciences, San Jose, CA, USA) equipped with CellQuest Pro™

4

software [4, 13] for the detection of red JC-1 aggregates (590 nm emission) or green

5JC-1 monomers (527 nm emission). The resulting data were evaluated using FlowJo

6version 7.6.3 software (TreeStar, San Carlos, CA), exported to Excel spreadsheets, and7subsequently analyzed using the statistical application R.

89

Measurement of total cellular O2 consumption rate (OCR)

10

Total OCR was measured as described previously [12, 16]. Briefly, SH-SY5Y

11

cells were trypsinized and suspended at a concentration of 1 × 107 cells/ml in

12

RPMI containing 10% FBS and 25 mM HEPES buffer. For each experiment,

13

equal numbers of cells (suspended in 1 ml) were pipetted into the chamber of an

14

Oxytherm electrode unit (Hansatech Instruments, Norfolk, UK), which uses a

15

Clark-type electrode to monitor the concentration of dissolved O2 in the sealed

16

chamber over time. The test reagents including lidocaine, rotenone and FCCP

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were added into the chambers immediately before each measurement. The

2

resulting data were exported to a computerized chart recorder (Oxygraph;3

Hansatech Instruments), which calculated the OCR values. The temperature was4maintained at 25°C during measurement. The concentrations of O2 in 1 ml of

5

DMEM medium lacking cells was also measured over time and utilized as the

6background. O2 consumption experiments were repeated at least three times, and7data were expressed as means ± SD [12].

89Live cell ROS imaging

10

For evaluation of intracellular ROS generation, control and lidocaine-treated

11

SH-SY5Y cells were treated with the ROS-sensitive dye 2',127'-dichlorodihydrofluorescin diacetate (DCFH-DA) and analyzed using a

13

BioStation IM live cell time-lapse imaging system (Nikon, Tokyo, Japan) at 37°C

14

and 5% CO2; phase-contrast and fluorescence images were acquired at 15 min

15intervals [18, 19].

16

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Statistical Analysis

2

All experiments were repeated at least twice and each sample was evaluated in3

triplicate. Representative data, expressed as means ± SD, are shown. Differences4

between results were evaluated by one-way analysis of variance (ANOVA) or

5

two-way ANOVA, followed by Dunnett’s test for multiple comparisons. All statistical

6

analyses were performed using EZR (Saitama Medical Center, Jichi Medical

7

University), which is a graphical user interface for R (The R Foundation for Statistical

8

Computing, version 3.1.3) [20]. More precisely, it is a modified version of R

9commander software (version 1.6–3) and includes statistical functions that are10

frequently used in biostatistics. P-values < 0.05 were considered statistically11significant. All graphs were generated by the application Prims 6 for Mac OS X

12

(GraphPad Software Inc., La Jolla, CA USA).

13
14
15

Results

16

Lidocaine induces SH-SY5Y cell death in a dose- and time-dependent manner

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1

MTS/PES2([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra

3

zolium, inner salt]/phenazine ethosulfate) assays were utilized to investigate the effects

4of lidocaine on neuronal cell proliferation or viability. In this assay, the NADPH and

5

NADH produced by dehydrogenase enzymes of metabolically active cells bioreduce the6

MTS tetrazolium compound (Owen’s reagent) into a colored formazan product that is

7

soluble in tissue culture medium. Treatment with 1 mM, 4 mM, and 10 mM lidocaine

8

significantly suppressed SH-SY5Y cell viability at 12 h, 24 h, and 48 h after9

administration in both a dose- and time-dependent manner (Fig. 1A). In contrast, 100

10µM lidocaine had no effect on cell viability (Fig. 1A). To elucidate the mechanism by

11which lidocaine suppresses cell viability, we evaluated the effect of this compound on

12

the levels of apoptosis in SH-SY5Y cells. Notably, treatment with 4 mM or 10 mM

13lidocaine resulted in significant increases in caspase-3/7 activation after 12 h, 24 h, and

1448 h (Fig. 1B), while treatment with 4 mM and10 mM lidocaine yielded statistically15

significant activation of caspase-9 (Fig. 1C), as well as increased levels of cleaved

16

PARP and caspase-9 (Fig. 1D), after 24 h.

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To confirm these findings, SH-SY5Y cells were stained with PI and recombinant

2

FITC-conjugated annexin V antibodies and evaluated by flow cytometry. Cells treated3with 4 mM or 10 mM lidocaine exhibited significantly increased numbers of PI-positive4

or annexin V-positive cells after 12 h and 24 h of treatment (Fig. 1E and Supplementary

5Fig. S1A–S1E). Additionally, treatment with 4 mM or 10 mM lidocaine resulted in

6

significant reductions in mitochondrial voltage (Fig. 1F and Supplementary Fig. 2A–

72D), while treatment with 10 mM lidocaine resulted in significant increases in

8

SH-SY5Y cell death after 12 h, as indicated by the levels of LDH release (Fig. 1G).

9
10

Critical involvement of mitochondria in lidocaine-induced cell death11

We next examined the effects of lidocaine treatment on HeLa cervical carcinoma cells, as

12well as the HeLa-derived cells lines EB8, which lack mitochondrial DNA (ρ0 cells), and

13

HeEB1, a hybrid clone of EB8 cells containing mtDNA from wild-type HeLa cells.

14

Compared to HeLa cells and HeEB1, EB8 cells exhibited lower levels of O2 consumption

15(Supplementary Fig. S3A) than the parental HeLa cells. Meanwhile, treatment with 4 mM

16

and 10 mM lidocaine resulted in significant increases in caspase-3/7 activation and cell

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death in both HeLa cells (Fig. 2A) and HeEB1 cells after 24 h, compared to the control

2

population (Supplementary Fig. S3B and S3C). Conversely, only 10 mM lidocaine

3

treatment induced caspase-3/7 activation in the EB8 ρ0 cell line (Fig. 2A). Notably,4

however, treatment with 10 mM lidocaine resulted in significantly higher levels of

5caspase-3/7 activity in HeLa cells than in EB8 cells. Moreover, flow cytometry analysis

6detected no significant difference in the levels of cell death between the untreated EB-8

7

ρ0 control population and the population subjected to 4 mM-lidocaine treatment,

8

indicating that, in contrast to the parental HeLa cell line, EB-8 cells are resistant to low

9levels of lidocaine (Fig. 2B). These results prompted us to examine the involvement of10

mitochondria in lidocaine-induced cell death. First, we investigated the effect of lidocaine

11

on oxygen consumption in SH-SY5Y cells; compared with the untreated control

12

population, SH-SY5Y cells treated with 4 mM and 10 mM lidocaine exhibited reduced

13

OCRs (Fig. 2C). We next evaluated the effects of mitochondrial ETC inhibitors, including

14

100nM rotenone, 2.5µg/ml oligomycin, and 4µM antimycin A, on SH-SY5Y cells. As

15

expected, each of these three reagents significantly suppressed MTS conversion (Fig. 2D).

16

Treatment with 1mM lidocaine or each ETC inhibitors did not elicit caspase 3/7 activation

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or ROS generation in SH-SY5Y cells (Figs. 2E and 2F). Notably co-treatment with 1mM

2

lidocaine and rotenone effectively induced the caspase activation. (Figs. 2E and 2F). In

3

this study, we used three types of ETC inhibitors. Although all the ETC inhibitors

4

increased caspase 3/7 activity and ROS generation, only rotenone exerted statistically5significant effect. Rotenone is a Complex I inhibitor. Oligomycin is an ATP synthase6

inhibitor. Antimycin A is a Complex III inhibitor. The evidence warrants further

7

investigation to clearly elucidate the target(s) of lidocaine and the mechanism of the8

synergistic effect of lidocaine and ETC inhibitors.

9

These findings are therefore consistent with those obtained by flow cytometry and10indicate that lidocaine promotes cell death by targeting mitochondria.

1112Effect of synthetic antioxidants on lidocaine-induced cell death13

Several reports have indicated that ROS play a critical role in mitochondria-dependent

14

cell death. As demonstrated in Fig. 3A, 4 mM and 10 mM lidocaine treatment induced

15

ROS accumulation in SH-SY5Y cells within 6 h. Notably, however, these increases in

16

ROS were blocked upon treatment with the antioxidant NAC (Fig. 3A). We therefore

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further examined the effects of the antioxidant NAC on SH-SY5Y cells. Treatment with2

10 mM NAC blocked the suppression of SH-SY5Y viability observed upon treatment3

with 4 mM lidocaine (Fig. 3B). Moreover, NAC inhibited the 4 mM lidocaine-induced4

increase in caspase-3/7 activity (Figs. 3C) in a dose-dependent manner. While neither 4

5

mM nor 10 mM NAC treatment had any significant effect on the suppression of cell

6

viability mediated by 10 mM lidocaine treatment (Fig. 3B), the levels of caspase-3/7

7

activation induced by 10 mM lidocaine were partially suppressed by exposure to 4 mM

8

and 10 mM NAC (Fig. 3C). Similar effects were observed in SH-SY5Y cells treated9with another antioxidant, Trolox (250 µM; Fig. 3F). 250µM Trolox suppressed ROS

10generation elicited by 4mM lidocaine (Supplementary S3E). 250µM Trolox treatment

11

significantly suppressed 4mM-lidocaine induced cell death but not 10mM-lidocaine

12

induced death (Fig. 3F, right panel). Although neither 4mM nor 10mM-lidocaine

13

induced caspase 3/7 activation was not statistically significant, 250µM Trolox

14

suppressed 4mM-lidocaine induced the caspase activation. Likewise, flow cytometry

15

analyses demonstrated the NAC treatment suppressed the effects of 4 mM and 10mM

16

lidocaine on cell death (Fig. 3E and Supplementary Fig. S1F–S1H), mitochondrial

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voltage (Supplementary Fig. S2E–S2G). LDH release by 10mM lidocaine was not

2

suppressed by NAC or Trolox treatment (Supplementary Fig.S3E).

3
4

Effects of an endogenous antioxidant on lidocaine-induced cell death5

Next, we chose to examine the effects of TRX, an endogenous redox active protein, on

6

lidocaine-induced cell death. First, however, we evaluated the effects of GGA, which is

7

known to be an inducer of TRX [21, 22], on SH-SY5Y cells. Consistent with the results

8

obtained using ROS scavengers, pre-treatment of SH-SY5Y cells with 5 µM and 10 µM

9GGA for 2 h resulted in reduced 4 mM lidocaine-induced cell death and caspase-3/7

10

activation (Fig. 4A–4C). 10µM GGA suppressed ROS generation elicited by 4mM

11lidocaine (Supplementary Fig. S3F). Even 10µM GGA did not inhibit cell death or LDH

12

release induced by 10mM lidocaine (Fig. 4B and Supplementary Fig. S3E).

13

Surprisingly, however, treatment with 10 µg/ml rhTRX failed to suppress

14

lidocaine-induced caspase-3/7 activation (Figs. 4D–4F).

15

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Effects of synthetic antioxidants on mepivacaine and bupivacaine-induced cell

2

death

3

Lastly, we examined the in vitro effects of other local anesthetics on SH-SY5Y cells.

4

Similar to those treated with 4 mM lidocaine, cells treated with 1 mM mepivacaine or 1

5mM bupivacaine exhibited significantly reduced cell viability and increased caspase-3/7

6

activation (Figs. 5A and 5B), and these effects were blocked by treatment with 10 mM

7

NAC or 10 µM GGA (Figs. 5A and 5B). These findings indicate that the8anesthetic-mediated increases in cell death observed in this study are not exclusive to

9lidocaine.

10
11
12

Discussion

13

In this study, we demonstrated that the local anesthetic lidocaine suppresses the14

mitochondrial ETC in neuronal SH-SY5Y cells in a dose- and time-dependent manner,

15

thereby attenuating mitochondrial membrane potential, inducing ROS production, and16

activating caspase-9- and caspase-3/7-mediated apoptosis and necrosis. Moreover, we

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observed similar effects in cells treated with mepivacaine and bupivacaine. Intriguingly2

treatment with the antioxidants NAC and Trolox successfully suppressed these effects

3

by scavenging the ROS derived from mitochondria.

45

Lidocaine induces two types of cell death

6

Multiple studies have reported that clinically relevant concentrations (500 µM to 24

7mM) of local anesthetics such as lidocaine are capable of inducing cell death in cells of

8

neuronal origin as well as in established cell lines derived from cancerous tissues [23,924]. Consistent with these findings, we demonstrated that 1 mM–10 mM lidocaine was10

sufficient to promote cell death in neuronal SH-SY5Y cells and HeLa cervical11

carcinoma cells (Fig. 1, Fig. 2, and Supplementary Fig. S1).

12
13

Lidocaine induces apoptosis and necrosis in a dose-dependent manner

14

There are at least two modes of cell death: apoptosis and necrosis [25]. Apoptosis is a15

strictly regulated (programmed) process involving the activation of specific cysteine

16

proteases that is responsible for the ordered removal of superfluous, aged, or damaged

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cells. Notably, while this process plays critical roles in both health and disease, necrosis

2

is solely the outcome of severe and acute injury. Apoptosis involves the regulated3

activity of catabolic enzymes (proteases and nucleases) within a near-to-intact plasma

4

membrane, and is commonly accompanied by characteristic changes in nuclear

5morphology and chromatin biochemistry.

6

In this study, lidocaine treatment promoted caspase-3/7 and caspase-9 activation, as

7

well as PARP cleavage, in SH-SY5Y cells (Fig. 1C and 1D). As such, these data

8

indicate that lidocaine induces cell death via an authentic apoptosis pathway.

9

Meanwhile, flow cytometry analyses demonstrated that treatment of cells with greater

10than 4 mM lidocaine especially 10mM lidocaine also resulted in increased numbers of

11

PI- and annexin V-positive cells (Supplementary Fig. S1). These data strongly suggest

12that lidocaine elicits both types of cell death in a dose-dependent manner in vitro.

13

Intriguingly, however, the observed increase in caspase 3/7 but not in cell death with

14

LDH release, was significantly suppressed by treatment with NAC, Trolox and GGA.

15

The evidence suggests that the antioxidants preferentially inhibit apoptosis rather than

16

necrosis.

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1
2

Functional mitochondria are necessary for lidocaine-induced apoptosis but not for3

necrosis4

Local anesthetics have been found to inhibit mitochondrial ETC at several points along5

the respiratory chain, and to inhibit F1-ATPase activity and adenine-nucleotide

6transport [26]. In this study, we also demonstrated the lidocaine inhibited oxygen

7

consumption by mitochondria (Fig. 2C). Moreover, using ρ0 cells lacking

8

mitochondrial DNA, we showed that functional mitochondria are required for

9lidocaine-induced cell death following caspase activation but not for lidocaine-induced

10

necrosis with LDH release (Figs. 2B and 2E). These data strongly suggests that11

mitochondria comprise a critical target for lidocaine-induced cell death, particularly for

12

apoptosis.

13

It was previously reported that dysfunction of the mitochondrial ETC due to mutations

14

in OXPHOS subunits or treatment with chemical inhibitors is generally associated with15

increased production of mitochondrial ROS, including superoxide anions, hydroxyl

16

radicals, and hydrogen peroxide [27]. Specifically, inhibitor studies using isolated

-26-

1

mitochondria demonstrated that complexes I and III of the ETC can act as relevant2sources of mitochondrial ROS [27]. Consistent with these findings, HEK293 cells

3

treated with rotenone and antimycin A for inhibition of complexes I and III,4

respectively, exhibited increased ROS production and induction of oxidative stress [28].

5

Meanwhile, ROS production was not observed in ρ0 cells in this study (data not

6

shown).

7
8

ROS derived from mitochondria promote lidocaine-induced apoptosis

9

Another intriguing finding presented in this report was that both lidocaine-induced

10

apoptosis and necrosis were ROS-dependent. The results presented in Fig. 3A clearly11demonstrate that lidocaine treatment induced ROS generation. And while the precise

12

origin of these ROS remains unclear, our findings strongly suggest that mitochondria

13

play a critical role in this process (Figs. 2B and 2C). As such, these data imply that14

lidocaine-mediated cell death is dependent on mitochondria. Consistent with this15

conclusion, the mitochondrial DNA-deficient ρ0 cells were resistant to lidocaine

16

treatment (Fig. 3B). Moreover, our data are consistent with a previous report

-27-

1

demonstrating that tetracaine-induced apoptosis in rat cortical astrocytes is associated2with increased ROS production [4].

34Effect of antioxidants on lidocaine-induced cell apoptosis

5

Oxidative stress in response to various external stimuli has been implicated in the6

induction of apoptosis. Specifically, oxygen free radicals induce DNA sequence

7changes and rearrangements that may trigger apoptotic cell death of neuronal cells. In

8this study, we provided the first evidence that ROS scavengers such as NAC and Trolox

9

can significantly suppress lidocaine-induced cell death in vitro. Conversely, while

10

treatment with rhTRX had no affect on apoptosis or necrosis, GGA, which is known to

11induce TRX expression and is itself an antioxidant, exerted a protective effect against

12

lidocaine-induced cell death. Although the molecular mechanisms underlying these13

discrepancies are unclear, it is possible that extracellular administration of rhTRX is an14

ineffective method for modulating intracellular redox status [22, 29, 30]. Also, GGA

15

has been shown to promote the expression of heat shock protein 70, which was reported

16

to alleviate cellular stress and exert cytoprotective effects [31, 32]. As such, the

-28-

1

observed beneficial effects of GGA on cell survival may be dependent on induction of

2

HSP70 and not TRX. In any case, future studies using cells that overexpress TRX may

3better elucidate whether this protein can inhibit anesthetic-induced cell death.

45Limitations

6

There are several limitations to the present study. First, the established SH-SY5Y and7

HeLa cell lines were used exclusively for all experiments. Although SH-SY5Y cells are8

derived from neuronal tissue and exhibit several characteristics similar to neurons, our9experimental results and conclusions cannot necessarily by extrapolated to neuronal

10

injuries induced by local anesthetics in an in vivo setting. Furthermore, although we

11evaluated the effects of lidocaine on ρ0 cells, which lack mitochondrial DNA, the12majority of the data were obtained using HeLa cells, primarily due to technical issues.

13

Lastly, the results presented in this study demonstrate that local anesthetics negatively

14

affect mitochondrial activity, thereby inducing apoptosis; however, the specific15molecular targets have yet to be identified. As such, the identification of such targets

16

comprises a critical goal for future studies.

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1
2

Conclusions

3

In this study, we demonstrated that the local anesthetics lidocaine, mepivacaine, and4bupivacaine induce two types of cell death in neuronal cells in vitro. In particular,

5treatment with 1–4 mM lidocaine promoted apoptosis, while treatment with 10 mM

6lidocaine induced cell death with LDH release. Moreover, our data demonstrate that

7

these compounds specifically target mitochondria, and that the ROS produced by

8

mitochondria play an integral role in the observed induction of apoptosis. Lastly, we

9demonstrated that scavenging of ROS with antioxidants such as NAC, Trolox, and10

GGA preserved mitochondrial voltage and prevented apoptosis by suppressing caspase11

activation.

12
13
14

List of Abbreviations

15

NAC: N-acetyl cysteine; ROS: reactive oxygen species; TRX: thioredoxin; OCR:

16

oxygen consumption rate

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1

Declarations

2

Ethics of approval and consent to participate

3

Not applicable

4Consent for publication

5

This manuscript does not contain any individual persons data, we state “Not applicable”

6in this section.

7

The authors agree to conditions of submission, BioMed Central's copyright and license

8agreement and article-processing charge (APC).

9
10

Availability of data and materials

11

The datasets analysed in the current study available from the corresponding author on

12

reasonable request.

13
14

Competing interests

15

The authors declare no competing interests.

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1
2

Funding3This work was supported by grants from the Japan Society for the Promotion of Science

4(KAKENHI; Grant #26670693 and #24592336 to K.H., and #25462457 to K.N.) and by

5

the research grant D2 from Kansai Medical University.

6
7

Author Contributions

8

AO, KS, and KH designed the study, performed the data analyses, and wrote the9manuscript. CS and KN collected and analyzed the data. MT, KO, and YM analyzed the10data. KT provided experimental materials. All authors read and approved the final

11

version of the manuscript.

12
13

Acknowledgements

14

We would like to Editage (www.editage.jp) for English language editing.

15

16

Additional files

-32-

1

Additional file: Supplementary Figure S1 Analysis of cell apoptosis by FACS

2

Levels of cell apoptosis were measured using an Annexin V-FITC Apoptosis

3

Detection Kit (BioVision, Milpitas, CA, USA), according to the manufacturer’s

4

instructions. For these analyses, SH-SY5Y cells were seeded into 6-well plates (35× 105 cells/well) and incubated overnight. The following day, cells were treated

6with the indicated concentrations of the appropriate drug(s) for varying lengths of

7time and harvested by centrifugation at 1,200 rpm for 3 min. The culture

8

supernatants were discharged, and the resulting pellets were resuspended in a

9

mixture comprised of 500 µl binding buffer, 5 µl Annexing V-FITC, and 5 µl

10propidium iodide (PI; 50 µg/ml) for 5 min at room temperature in the dark and11analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA,

12USA). (PDF 2.1 MB)

13
14

Additional file: Supplementary Figure S2 Mitochondrial membrane potential

15

(ΔΨm)

-33-

1

Mitochondrial membrane potential was determined by flow cytometry using a2

MitoPT™ JC-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN,

3USA), according to the manufacturer’s instructions. For these analyses, SH-SY5Y

4cells were seeded into 6-well plates (3 × 105 cells/well) and cultivated overnight.

5

The following day, cells were treated with the indicated concentrations of the

6

appropriate drug(s) for varying lengths of time and then pelleted by centrifugation

7

at 1,200 rpm for 3 min. Supernatants were discharged, and cells were resuspended8

in JC-1, incubated at 37°C for 15 min in the dark, and collected by centrifugation9

at 1,200 rpm for 3 min. Supernatants were again discharged and the remaining cell

10

residues were suspended in 500 µl assay buffer. Samples were subsequently

11

analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA,

12USA) equipped with CellQuest Pro™ software for the detection of red JC-1

13

aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). (PDF

14

2.1 MB)

15
16

Additional file: Supplementary Figure S3 Results of HeLa cell-derivatives EB8 and

-34-

1

HeEB1

2

(A) Oxygen consumption rate of HeLa cells, EB8 and HeEB1cells were demonstrated.

3(B) Graphic depiction of reactive oxygen species (ROS) production in HeLa cells and4EB8 cells exposed to the indicated concentrations of lidocaine (0, 4, or 10 mM) for 6 h

5(n = 3). Data depict the ratio of ROS production in treated cells compared to that in the6untreated control group (HeLa cells).

7(C) Activities of Caspase3/7 of HeLa cells and HeEB1 cells were demonstrated. (D)8Levels of cell death were measured using an Annexin V-FITC Apoptosis Detection Kit

9evaluated by FACS were demonstrated. (E) Graphic depiction of the levels of cell death

10

among treated and untreated cell populations. Cell death was evaluated by measuring

11

the levels of lactate dehydrogenase (LDH) within culture supernatants (n = 3) in the

12presence or absence of 10 mM N-acetyl cysteine (NAC), 250µM Trolox and 10µM

13GGA. Control is LDH activity treated by lysis buffer. (F) Graphic depiction of reactive

14

oxygen species (ROS) production in SH-SY5Y cells exposed to 4 mM) for 6 h (n = 3)

15

in the presence or absence of 10 mM N-acetyl cysteine (NAC), 250µM Trolox and

16

10µM GGA. Data depict the ratio of ROS production in treated cells compared to that

-35-

1

in the untreated control group. Data presented in A–E expressed as means ± standard

2

deviations (SD). #p < 0.05 compared with the control cell population at the same time3

period. (PDF 602 KB)

4

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Figure legends

16

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1

Figure 1. Lidocaine induces SH-SY5Y cell death in a dose- and time-dependent2

manner. Neuronal SH-SY5Y cells were exposed to the indicated concentrations (0.1, 1,

3

4, or 10 mM) of lidocaine for varying lengths of time (0, 12, 24, and 48 h). (A) Graphic

4

depiction of the levels of cell viability of treated and untreated cells at each time point,5

as evaluated by MTS

6[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz

7

olium] assay analysis (n = 4). (B and C) Graphic depictions of caspase-3/7 (n = 3) and8

caspase-9 (n = 5) activity in each treatment group at different time points, as determined

9

using an Apo-ONE™ Homogeneous Caspase-3/7 Assay Kit and a Caspase-Glo™ 9

10

Assays Kit, respectively. (D) Immunoblot analysis of the levels of poly (ADP-ribose)

11

polymerase (PARP), cleaved caspase-9 and β-actin in the lysates of treated and

12untreated cells after 24 h. The blots are derived from two independent experiments. (E)13Treated and untreated cells were harvested, and the levels of cell death were analyzed

14

by flow cytometry. The ratio of propidium iodide (PI)-positive and/or annexin

15

V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the16

percentage of dead cells (Supplemental Fig. S1) (n = 3). (F) Graphic depiction of the

-43-

1

average mitochondrial membrane potential (ΔΨm) of treated and untreated cells (n = 3)2

at each time point, as measured using a MitoPT™ JC-1 Assay Kit. Values indicate the3

ratio [Q2/(Q2+Q4)] of green JC-1 monomers (527 nm emission) to red aggregates (5904nm emission). (G) Graphic depiction of the levels of cell death among treated and5untreated cell populations. Cell death was evaluated by measuring the levels of lactate

6dehydrogenase (LDH) within culture supernatants (n = 4). Control is treatment by lysis

7

buffer. Data presented in A–C and E–G are expressed as means ± standard deviations

8(SD). Differences between results were evaluated by two-way ANOVA (A, B, E and F),

9followed by Dunnett’s test for multiple comparisons in each group and one-way

10

ANOVA (C and G) followed by Dunnett’s test for multiple comparisons. *p < 0.05

11

compared with the control cell population at incubation time 0 h (no treatment). #p <12

0.05 compared with the control cell population at the same time period (group).

13
14

Figure 2. Critical involvement of mitochondria in lidocaine-induced cell death.

15

HeLa cells and EB-1 cells (HeLa cells lacking mitochondrial DNA) were exposed to the16

indicated concentrations of lidocaine (0, 0.1, 1, 4, or 10 mM) for 24 h. (A) Graphic

-44-

1

depiction of the levels of caspase-3/7 activity in each treatment group, as determined2

using an Apo-ONE Homogeneous Caspase-3/7 Assay Kit™ (n = 4). (B) Treated and3

untreated cells were harvested, and the levels of cell death were analyzed by flow

4

cytometry (n = 4). The ratio with propidium iodide (PI)- or annexin V-positive cells

5[(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] were indicated as dead cells (Supplemental

6

Fig. S1). (C) Graphic depiction of the oxygen consumption rate (OCR) in untreated

7

SH-SY5Y cells and cells treated with lidocaine (0.1, 1, 4, or 10 mM), rotenone (1008nM), or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (50 nM). Values

9

are presented as ratios of OCR compared to that in the control (without lidocaine

10

treatment) group (n = 4). (D and E) Graphic depiction of the levels of cell viability

11

among SH-SY5Y cells treated with mitochondrial ETC inhibitors. Cells were treated12with 1 mM lidocaine and either 100n M rotenone, 2.5µg/ml oligomycin, or 4µM

13antimycin A, and subjected to (D) MTS

14[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz

15

olium] assay (n = 3) or (E) caspase-3/7 activity assay (n = 3) analysis. (F) Graphic

16

depiction of reactive oxygen species (ROS) production in SH-SY5Y cells exposed to

-45-

1

1m lidocaine in the presence or absence of 100nM rotenone, 2.5µg/ml oligomycin and

2

4µM antimycin A for 6 h (n = 3). Data depict the ratio of ROS production in treated3

cells compared to that in the untreated control group. All data were expressed as means

4± standard deviations (SD). . Differences between results were evaluated by two-way

5

analysis of variance (ANOVA) (A, B, E and F) followed by Dunnett’s for multiple

6

comparisons in each group or one-way ANOVA (C and D), followed by Dunnett’s test

7

for multiple comparisons. #p < 0.05 compared with the control treatment population in8

the same group.

910

Figure 3. Effect of synthetic antioxidants on lidocaine-induced cell death. (A)11Graphic depiction of reactive oxygen species (ROS) production in SH-SY5Y cells

12

exposed to the indicated concentrations of lidocaine (0, 0.1, 4, or 10 mM) for 6 h (n = 3)

13

in the presence or absence of 10 mM N-acetyl cysteine (NAC). Data depict the ratio of

14

ROS production in treated cells compared to that in the untreated control group. (B–E)

15

SH-SY5Y cells were exposed to the indicated concentrations of lidocaine (0, 4, or 10

16mM) for 24 h in the presence or absence of NAC (4 or 10 mM). (B) Cell viability and

-46-

1

(C) caspase-3/7 activity were evaluated by MTS

2[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz

3olium] assay (n = 3) and Apo-ONE™ Homogeneous Caspase-3/7 Assay (n = 3)

4

analysis, respectively. (D) Cells were harvested and lysates were subjected to5

immunoblot assay analysis using antibodies specific to poly (ADP-ribose) polymerase

6(PARP) and cleaved caspase-9. (E) Graphic depiction of the levels of cell death among

7

treated and untreated cell populations, as evaluated by flow cytometry (n = 4). The ratio

8with PI or annexin V positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] were

9indicated as dead cells (Supplemental Fig. S1). (F) SH-SY5Y cells were exposed to the10indicated concentrations of lidocaine (0.1, 1, 4, or 10 mM) in the presence or absence of11

250 µM Trolox for 24 h, and subjected to caspase-3/7 activity assay analysis (n = 4)

12(left panel). Graphic depiction of the levels of cell death among treated and untreated

13cell populations, as evaluated by flow cytometry (n = 3) (right panel). The ratio with PI

14

or annexin V positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] were indicated as

15

dead cells. Differences between results were evaluated by one-way analysis of variance

16(ANOVA) (A) followed by Dunnett’s test for multiple comparisons or two-way

-47-

1

ANOVA (B, C, E and F) followed by Dunnett’s test for multiple comparisons in each2

group. *p < 0.05 compared with the control cell population at 0 h (no treatment).

30.05 compared with the control treatment population in the same group.

#p <

4
5

Figure 4. Effects of an endogenous antioxidant on lidocaine-induced cell death. (A–6C) SH-SY5Y cells were exposed to the indicated concentrations of lidocaine (0, 4, or 10

7mM) for 24 h in the presence or absence of pretreatment with 5 or 10 µM teprenone

8(geranylgeranylacetone, GGA) for 24 h. (A) Cell viability and (B) caspase-3/7 activity

9were evaluated by MTS

10[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz

11

olium] and Apo-ONE™ Homogeneous Caspase-3/7 Assay analysis, respectively (n = 3

12

for each). (C) Graphic depiction of the levels of cell death among treated and untreated

13cell populations, as evaluated by flow cytometry (n = 4). The ratio with PI or annexin V

14positive cells were indicated as dead cells. (D–F) SH-SY5Y cells were exposed to the15

indicated concentrations of lidocaine (0, 1, 4, or 10 mM) for 24 h in the presence or16absence of pretreatment with 10 µM recombinant human thioredoxin (TRX) for 2 h.

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1

Graphic depictions of (D) cell viability (n = 3), (E) caspase-3/7 activity (n = 3), and (F)2cell death (n = 4), as determined by MTS, Apo-ONE™ Homogeneous Caspase-3/7, and3flow cytometry analysis, respectively. All data were expressed as means ± standard

4

deviations (SD). Differences between results were evaluated by two-way analysis of

5variance (ANOVA) followed by Dunnett’s test for multiple comparisons in each group.

6*p < 0.05 compared with the control cell population at time 0 h (no treatment). #p <7

0.05 compared with the control treatment population in the same group.

8
9

Figure 5. SH-SY5Y cells were exposed to the indicated concentrations of local

10

anesthetics in the presence or absence of the indicated concentrations of 10 mM N-acetyl

11cysteine (NAC) and 10 µM geranylgeranylacetone (GGA) for 24 h. (A) Levels of cell

12

viability and (B) caspase-3/7 activity were evaluated by MTS

13[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraz

14

olium] and Apo-ONE™ Homogeneous Caspase-3/7 Assay analyses (n = 3 for each).

15

Data were expressed as means ± standard deviations (SD). Differences between results

16

were evaluated by two-way analysis of variance (ANOVA) followed by Dunnett’s test

-49-

1

for multiple comparisons. *p < 0.05 compared with the control cell population at time 02

h (no treatment). #p < 0.05 compared with the control treatment population in the same

3

group

-50-

1
AFigure(%)

# # *
#
0h

*

# # *
#

12 h

48 h

#
cont.
lido-100µM
lido-1mM
lido-4mM
lido-10mM

Caspase 3/7
activity

#

*

*

*
#

#

24 h

48 h

#

0h

12 h

G

(%)

D

IB : PARP
IB : c-Caspase-9
IB : β-actin

-

1

4

10

-

#

12 h

24 h

(%)

lidocaine(mM)
Lidocaine (mM)##cont. 0hcont.
lido-100µM
lido-1mM
lido-4mM
lido-10mM##

LDH activity

Caspase 9
activity

#

24 h

(%)

*

#

*

C

12 h

F

(%)

#

#

#

cont.
lido-100µM
lido-1mM
lido-4mM
lido-10mM

# # *
#

24 h

*

#

ΔΨm
supression ratio

B

*

* *

* *

#

cont.
lido-100µM
lido-1mM
lido-4mM
lido-10mM

cell death

Cell Viability*

Click here to download Figure NAC-fig1-R3.eps

(%)

E

1

4

10
PARP
cleaved PARP

#

0

0.1 1

4

10 lysis B

cont.
lido-100µM
lido-1mM
lido-4mM
lido-10mM

A

Click here to download Figure NAC-fig2-R3.eps

D
(%)

(%)

#

Cell Viability

Caspase 3/7
activity##

lidocaine-1mM
Celllidocaine (mM)HeLa
0 1 4 10 0

+

# #

#

+

+

+

cont.
cont.
rotenone(100nM)
rotenoneoligomycin(2.5µg/mL)
oligomycin
antimycinA(4µM)
antimycinAEB8
1 4 10

E

B

(%)

(%)

#
#

Cell
lidocaine (mM)

C

#

HeLa
0 1 4 10cont.
rotenone (100nM)
oligomycin (2.5µg/mL)
antimycinA (4µM)#
Caspase 3/7
activitycell death

EB8
0 1 4 10lidocaine-1mM

F(%)+ + + +

-

-

-

-

(%)

#

#

cont.
rotenone (100nM)
oligomycin (2.5µg/mL)
antimycinA (4µM)

#
ROS

OCR

Figure 2## #lidocaine (mM) 0 0.1 1
rotenone (nM)
FCCP (nM)

4 10lidocaine-1mM
100
50+ + + +

-

-

-

-

FigureA3

Click here to download Figure NAC-fig3-R3.eps

D

(%)

24Hr

#

ROS

#

NAC (mM)

-

-

10

10

-

10

Lidocaine (mM)

0

4

4

0

10

10
PARP
cleaved PARP

IB : PARP
IB : c-Caspase-9
lidocaine (mM) 0 0.1 1

4 10 0.1 1

IB : β-actin

4 10

NAC 10mM

E

B

(%)

(%)

#

lidocaine (mM)

0

4

10

cont.
NAC 4mM
NAC 10mM#
cell death

Cell Viabilitycont.
NAC 4mM
NAC 10mM##

lidocaine (mM)

0

4

10

F

C
(%)

# #

*
# #

0

4

10

trolox

#

#
#
#

-

-

cell deathcont.
NAC 4mM
NAC 10mM

Caspase 3/7
activity

Caspase 3/7
activity*

lidocaine (mM)

(%)

(%)

#

#

-

+ + +

-

-

+ + +

lidocaine (mM) 0 4 10

0 4 100 4 100 4 10-A(%) Click here to download Figure NAC-fig4-R3.eps

Dcont.
GGA 5µM
GGA 10µM#

Cell Viability

Cell Viability

(%)

*
*

lidocaine (mM)

0

4

#

lidocaine (mM)

10

# #
#

# #

0

1

4 10

0

1

4 10

rhTRX10µM

E(%)
Caspase 3/7
activitycont.
GGA 5µM
GGA 10µM#*#

lidocaine (mM)

0

#

4

(%)

#

Caspase 3/7
activityB##lidocaine (mM)

10

0

1

#

4 10

0

1

4 10

rhTRX10µM
(%)

F
cont.
GGA 5µM
GGA 10µM

*
#

*

(%)

#
cell deathCcell death

Figure 4###0 4 100 4 10#lidocaine (mM)

0

4

10

lidocaine (mM)rhTRX10µM

Click here to download Figure NAC-fig5-R3.eps

A

(%)

# # #

lidocaine (mM) 4 4 4 0
mepivacaine (mM)bupivacaine (mM)

1 1 1 0# # #Cell Viability

# # #

NAC
GGA

+

1 1 1 0
+

+

+

+

+

# # #

# # #

# # #

lidocaine (mM) 4 4 4 0
mepivacaine (mM)bupivacaine (mM)

1 1 1 0B

Caspase 3/7
activity

Figure 5

NAC
GGA

(%)

+

1 1 1 0
+

+

+
+

+

B

A
Q1 Q2

Q3 Q4
controlClidcocaine:100µM

Dlidcocaine:1mM

lidcocaine:4mMFE

lidcocaine:1mM
NAC:10mM

lidcocaine:10mM

G

H

lidcocaine:4mM
NAC:10mM

Okamoto Supplement S1

lidcocaine:10mM
NAC:10mM

A

B

controllidcocaine:1mMDC

lidcocaine:4mMEG

lidcocaine:10mM

F

lidcocaine:1mM
NAC:10mM

lidcocaine:10mM
NAC:10mM

Okamoto Supplement S2

lidcocaine:4mM
NAC:10mM

A

D

(%)

#

#

#

Cell Death

OCR

(%)

#

#
#

lidocaine (mM)

0

HeLa EB8 HeEB1

1

1

4 10 0

HeLa

B

E

4 10

HeEB1

(%)

ROS

LDH activity(%)

lidocaine (mM)

0 4 10 0
HeLa

lidocaine
(mM)

4 10
EB8

C

0 10 10 10 10 lysis B
NAC
Trolox
GGA

F
(%)

#

Caspase 3/7
activity
lidocaine (mM)

(%)

#

#

#

#

#

#

0

1

4 10 0

HeLa

Okamoto Supplement S3

1

4 10

HeEB1

ROS

#

lidocaine
(mM)

0

4

4 4 4
NAC
Trolox
GGA