R E S E A R CH AR T I C L E

NF-jB/p53-activated inflammatory response involvesin diquat-induced mitochondrial dysfunction and apoptosis

Su Eun Choi1,2 | Yun Sun Park1,2 | Hyun Chul Koh1,2,3

1Department of Pharmacology, College of

Medicine, Hanyang University, Seoul,

Republic of Korea

2Graduate School of Biomedical Science and

Engineering, Hanyang University, Seoul,

Republic of Korea

3Hanyang Biomedical Research Institute,

Seoul, Republic of Korea

Correspondence

Hyun Chul Koh, Department of

Pharmacology, College of Medicine,

Hanyang University, Sungdong-Gu, Hean-

dang-Dong 17, 133-791 Seoul, Republic of

Korea.

Email: hckoh@hanyang.ac.kr

Funding information

Korea Science and Engineering Foundation

through the Medical Research Center at

Hanyang University College of Medicine,

Republic of Korea, Grant Number: NRF-

2008-0062287; Basic Science Research

Program through the National Research

Foundation of Korea (NRF) (Ministry of

Education, Science and Technology), Grant

Number: 2015R1D1A1A01059531

AbstractInflammation generated by environmental toxicants including pesticides could be one of the fac-

tors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated

the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated

with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of cas-

pases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP

level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by

diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of

PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant

N-acetyl-cystein attenuated the cytotoxicity of caspase-3 pathways. These results demonstrate

that diquat-induced apoptosis is involved in mitochondrial dysfunction through production of ROS.

Furthermore, diquat increased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-

a (TNF-a) via inflammatory stimulation. Diquat induced nuclear accumulation of NF-jB and p53

proteins. Importantly, an inhibitor of NF-jB nuclear translocation blocked the increase of p53.

Both NF-jB and p53 inhibitors also blocked the diquat-induced inflammatory response. Pretreat-

ment of cells with meloxicam, a COX-2 inhibitor, also blocked apoptosis and mitochondrial

dysfunction. These results represent a unique molecular characterization of diquat-induced cyto-

toxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through

inflammatory responses via NF-jB-mediated p53 signaling. This suggests the potential to generate

mitochondrial damage via inflammatory responses and inflammatory stimulation-related neurode-

generative disease.

K E YWORD S

diquat, inflammatory response, mitochondrial dysfunction, NF-jB signaling, p53 signaling

1 | INTRODUCTION

Accumulating evidence suggests that environmental toxins, coupled

with genetic predispositions, are major contributing factors to the

development of neurodegenerative disease.1 Exposure to agricultural

chemicals in a rural environment, drinking well-water, and occupational

exposure have all been postulated to be environmental risk factors for

disease.2 Increasing evidence suggests an important role for exposure

to pesticides such as rotenone and paraquat in the pathogenesis of

neurodegenerative disease.3,4 Diquat is a redox-cycling compound

used as an aquatic herbicide, and both diquat and paraquat belong to

the bipyridyl group of herbicides. Diquat has replaced paraquat which

has been prohibited due to a suggested association with Parkinson’s

disease (PD), and is now in widespread use as an agricultural and home

use herbicide.5 Mitochondrial dysfunction has been implicated in neu-

ronal cell death, partly through increased ROS generation. Specifically,

mitochondrial complex I activity is selectively lost in the dopaminergic

neurons of patient with PD, whereas other electron-transport com-

plexes remain unchanged.6 The pesticides rotenone, paraquat, and

chlorpyrifos have been widely used in vivo and in vitro models of PD

due to their capacity to inhibit mitochondrial complex I activity in dopa-

minergic neurons.7–10Su Eun Choi and Yun Sun Park contributed equally to the work.

Environmental Toxicology. 2018;1–14. wileyonlinelibrary.com/journal/tox VC 2018Wiley Periodicals, Inc. | 1

Received: 18 September 2017 | Revised: 8 February 2018 | Accepted: 11 February 2018

DOI: 10.1002/tox.22552

ROS play a critical role in the regulation of physiological cellular

functions and are involved in pathologic conditions such as inflamma-

tion and cell death.11,12 Upregulated ROS generation results in calcium

dysregulation that leads to mitochondrial dysfunction and ultimately

results in the activation of apoptotic cascades and promotes inflamma-

tory processes via nuclear factor (NF)-jB activation.13–15 NF-jB

regulates transcription of genes involved in immune responses, inflam-

mation, cell differentiation, proliferation, and apoptosis.16,17 NF-jB,

which has been reported to increase in dopaminergic neurons of PD,

plays a prominent role in 6-OHDA-induced SH-SY5Y cell death.18

Inflammation is a tissue response to injury, ischemia, autoimmunity,

toxic metabolites, or infectious agents. It is closely related to the initia-

tion and progression of neuronal cell damage and plays a significant

pathological role in the development of neurodegenerative dis-

eases.19,20 p53 should be induced in inflammatory conditions, but not

under normal circumstances. Most cells have mechanisms to suppress

p53 when NF-jB is activated in order to favor cellular transformation

processes.21 Through various mechanisms, NF-jB can cripple cellular

p53 activation.22,23 NF-jB activation has also been suggested to have

both proapoptotic and antiapoptotic roles24,25 since it can activate cell

death genes such as p53 as well as pro-survival genes such as Bcl-

2.26,27 Therefore, we speculate that an NF-jB-regulated p53 pathway

might contribute to inflammatory responses and cell death triggered by

diquat.

It is therefore important to study the exact mechanisms involved

in diquat toxicity. The present investigation was designed to identify

pathways involved in diquat-induced apoptotic cell death in cultured

PC12 cells, a rat pheochromocytoma cell line used as a model of dopa-

minergic neurons, that produces catecholamines including DA. In addi-

tion, we investigated if diquat-induced inflammatory responses were

associated with alterations in transcription factors, and explored the

molecular mechanisms underlying mitochondrial dysfunction and cell

death induced by diquat.

2 | MATERIALS AND METHODS

2.1 | Cell culture and treatment

PC-12 cells were obtained from the American Type Culture Collection

(ATCC, VA) and cultured in RPMI-1640 medium supplemented with

10% heat-inactivated horse serum, 5% inactivated fetal bovine serum

(FBS), and 1% penicillin-streptomycin. Cells were seeded onto poly-L-

lysine-coated plates and grown at 378C in a humidified 5% CO2 atmos-

phere. Cells used for western blot analysis were grown in 100mm cul-

ture dishes, whereas those used for cell viability assays were grown in

96-well plates. Cells were plated at a density of 4 3 104 cells (96-well

plate) and allowed to attach overnight. Culture media were replaced

every 3 days. The cells were used for experiments prior to passage 15.

2.2 | Drug treatment

Diquat was obtained from Sigma–Aldrich (MO). A 40 mM diquat stock

was used to make the dilutions for cell treatment. The incubation time

for diquat (0–40 lM) treatment ranged from 0 to 24 h, as indicated in

the figures. Antibodies were used as follows: anti-COX-2 from Abcam

(Cambridge, UK); anti-p-NFjB, IkB, p53, Lamin B, cleaved caspase-3,

cleaved caspase-9, Bcl-2, Bax, COXIV, and b-actin from Cell Signaling

Technology (MA). TNF-a and cytochrome c antibodies were obtained

from Bio Vision Technology (CA). Selective inhibitors were pifithrin-a

from TOCRIS Bioscience (Bristol, UK) and SN50 from Calbiochem

(EMD Biosciences, Inc. San Diego, CA, USA). All other chemicals were

obtained from Sigma-Aldrich. SN50 and pifithrin-a were dissolved in

dimethyl sulfoxide (DMSO), which served as the vehicle control for

these agents. SN50 and pifithrin-a were administered 1h before diquat

exposure.

2.3 | Cell viability and cytotoxicity

Cell viability was measured by MTS assay (CellTiter96sRAQueous One

Solution Cell Proliferation Assay, Promega, WI, USA). Briefly, MTS was

added to PC12 cells in 96-well plates and the plates were incubated at

378C for 1 h in a humidified 5% CO2 atmosphere. Metabolically active

cells convert the yellow MTS tetrazolium compound to a purple forma-

zan product. The latter is soluble in tissue culture medium and the

quantity of formazan product as measured by absorbance at 450 nm is

directly proportional to the number of living cells in culture. Results are

expressed as cell number for each experimental condition. A lactate

dehydrogenase (LDH) cytotoxicity detection kit (Takara, MK401, Japan)

was used to measure the leakage of soluble cytoplasmic LDH into the

extracellular medium due to cell death. LDH converts pyruvate to lactic

acid in the presence of reduced b-nicotinamide adenine dinucleotide

(NADH), and any pyruvate not converted to lactic acid produces a

highly colored phenylhydrazone when treated with 2,4-dinitrophenyl-

hydrazine. After incubation in the presence of either diquat or vehicle,

culture medium was collected and centrifuged at 4 000 rpm for 10 min

at 48C. The LDH activity in the culture supernatant was measured after

transferring the supernatant to 96-well plates. The reaction was run in

the dark for 10 min prior to measurement, and the absorbance was

measured with a microplate reader at 490 nm.

2.4 | Measurement of intracellular ROS

Conversion of non-fluorescent chloromethyl-DCF-DA (2,7-dichloro-

fluorescindiacetate) to fluorescent DCF was used to monitor intracellu-

lar ROS production. Cells plated in coated 24-well plates were grown

in RPMI-1640 medium and treated with 20 lM diquat or PBS as a con-

trol for 4 h, with or without pre-treatment with the antioxidant, N-ace-

tyl cysteine (NAC). Because 24-h incubation with diquat leads to

significant reduction in cell density at higher concentration, ROS pro-

duction varies in a diquat concentration-dependent manner, and ROS

have a short half-life, cells were incubated with various concentrations

of diquat for 4 h. The medium was removed and cells were washed

with PBS. Then, 200 mL DCF-DA (10 lM) was added, followed by incu-

bation for 1 h at 378C in the dark and subsequent washing with PBS to

remove excess dye. Cells were detached with trypsin and washed in

PBS. After centrifugation, cell pellets were suspended in 500 mL PBS.

2 | CHOI ET AL.

Intracellular ROS production was detected by the signal obtained using

flow cytometry (BD FACS Calibur; BD bioscience; CA; Cell Quest Soft-

ware), and fluorescent images were acquired with an Olympus

microscope.

2.5 | Nuclear morphology assessment by fluorescence

microscopy

Nuclear morphological changes were measured using Hoechst

33342 (Invitrogen, Carlsbad, CA). Cells were grown on cover slips in

24-well plates coated with poly-L-lysine. After treatment with diquat

for 24 h, cellular monolayers in 24-well plates were fixed with 4%

paraformaldehyde for 20 min and stained with 5 lg mL21 Hoechst

33342 solution in the dark for 30 min at 378C. After washing with

PBS, morphological features of apoptosis such as cellular nucleus

shrinkage, chromatin condensation, intense fluorescence, and nuclear

fragmentation were monitored by fluorescence microscopy. Quanti-

tative evaluation of apoptotic cells was performed counting at least

2 000 cells in randomly chosen fields under a fluorescence micro-

scope after Hoechst labeling. Cells with condensed or fragmented

nuclei were scored as apoptotic, while those with uniformly stained

nuclei were scored as healthy.

2.6 | Mitochondrial complex I enzyme activity assay

Complex I activity was measured by monitoring the activity of NADH

dehydrogenase (OXPHOS complex I) in 96-well format (Mitosciences,

Eugene, OR). Briefly, crude mitochondria samples were prepared as for

the cytochrome c release assay and were adjusted to a sample concen-

tration of 5.5 mg mL21 in PBS. A 1/10 volume of detergent was added

to the samples, which were incubated for 30 min on ice to allow solubi-

lization. The mixture was centrifuged for 20 min at 12 000g and the

supernatant was collected. Prepared samples were adjusted to 200 mg

with incubation buffer, loaded on a plate, and allowed to incubate for

3 h at room temperature. Finally, the wells were washed twice with

buffer, and 200 mL of assay solution was added to each well. NADH

oxidation was monitored by measuring the decrease in absorbance at

450 nm in kinetic mode at room temperature for 30 min. The rate was

linear during this period. The levels of activity were the results of the

absorbance value normalized by protein concentration. RPMI-1640

and buffer vehicles were found not to affect complex I activity.

2.7 | ATP determination

ATP was measured through a bioluminescence assay using an ATP

determination kit (Molecular Probes, Eugene, OR) as described previ-

ously.28 PC12 cells survived until 4 h after 20 mM diqaut treatment and

began to die at 5 h. To determine ATP levels, diquat was treated for up

to 4 h. The cells were then resuspended in reaction buffer containing

1 mM dithiothreitol (DTT), 0.5 mM D-luciferin, and 12.5 lg mL21 firefly

luciferase. After a 15-min incubation, cellular ATP was analyzed using a

microplate luminometer (Panomics, Fremont, CA).

2.8 | Quantification of DA

The levels of DA were determined by a modified method.29 Cells in

100-mm culture dishes were washed three times with PBS and then

collected. Cells were homogenized in 0.2 N perchloric acid (PCA) con-

taining 0.1 M EDTA with an internal standard, centrifuged at

13,000 rpm for 10 min, and filtered through minispin filters for an addi-

tional 5 min at 13 000 rpm. Samples (culture media and cell lysate)

were subjected to alumina extraction and eluted in 0.1 N PCA. Samples

(20 mL) were injected with a Rheodyne injector for separation on a

reverse phase l-Bondapak C18 column (150 3 3.0 mm2, Eicom, Japan)

maintained at 308C with a column heater (Waters, Cotland, NY). The

mobile phase consisted of 0.05 M citric acid, 0.05 M disodium phos-

phate (pH 3.1), 3.2 mM 1-octanesulfonic acid (sodium salts), 0.3 mM

EDTA, and 12% methanol pumped at a flow rate of 0.5 mL min21 with

a Waters solvent delivery system. Electroactive compounds were ana-

lyzed at 1700 mV using an analytical cell with an amperometric detec-

tor (Eicom, Model ECD-300), Japan). Elution peaks were processed

using DS Chromatographic Software (Donam, Korea). DA and its

metabolite peaks were normalized by comparison to the respective

internal standard, and concentrations were calculated from an external

standard injected immediately before and after each experiment. The

pellet was dissolved in 1 m‘ 0.5 N NaOH. Protein content was deter-

mined using a DC protein assay kit (Bio-Rad). For further analysis,

amounts of intracellular DA were corrected for sample protein concen-

tration and expressed as ng mg21 protein.

2.9 | Western blot analysis

To determine levels of protein expression, extracts were prepared from

PC12 cells. Adherent cells were scraped off culture dishes and lysed by

incubation with radio-immunoprecipitation assay (RIPA) lysis buffer

(50 mM tris-HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1%

NP-40, 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride

(PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail

(Roche, IN) on ice. Collected cells were broken by sonication on ice and

centrifuged at 10 000g for 20 min at 48C. Protein concentrations were

determined with Bradford reagent, and 30 mg samples of extracted pro-

tein were resolved on SDS-polyacrylamide gels and then transferred to

nitrocellulose membranes. Membranes were incubated in the presence

of different primary antibodies at 48C overnight and then incubated

with secondary antibody coupled to horseradish peroxidase. Immuno-

reactivity was visualized using enhanced chemiluminescence (Amer-

sham, Buckinghamshire, England, UK). Protein bands were quantified

with a densitometer (Molecular Devices, VERSAmax, CA).

2.10 | Cell fractionation

Cells were lysed in buffer A (0.25 M sucrose, 10 mM Tris-HCl (pH7.5),

10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and

0.1 mM PMSF) with a homogenizer. Homogenates were centrifuged at

750g for 10 min at 48C and supernatants were collected and centri-

fuged at 10 000g for 20 min at 48C. The supernatants were used as the

cytosolic fraction, and pellets were used as the mitochondrial fraction.

CHOI ET AL. | 3

Pellets were resuspended in buffer B (0.25 M sucrose, 10 mM Tris-HCl

(pH7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol,

0.1 mM PMSF, and 1% NP40).

To prepare nuclear extracts, cells were washed twice with cold

PBS and detached from plates with detaching buffer (150 mM NaCl,

1 mM EDTA (pH 8.0), 40 mM Tris-HCl (pH 7.6)) for 5 min at room tem-

perature. The cells were transferred to microcentrifuge tubes and cen-

trifuged at 300g for 4 min at 48C. Supernatants were discarded, and

pellets were resuspended in 400 m‘ cold lysis solution A (10 mM Hepes

(pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) and

incubated on ice for 15 min. After addition of 10 mL of 10% Nonidet P-

40, the mixtures were vortexed briefly. Nuclei were then pelleted by

centrifugation at 2 800g for 4 min at 48C and resuspended in 50 mL of

ice-cold lysis solution B (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM

EDTA, 1 mM DTT, 1 mM PMSF). The mixtures were vigorously shaken

for 15 min at 48C, centrifuged at 15 000g for 5 min, and supernatants

were collected.

2.11 | Data analysis

The data represent results from at least three independent experi-

ments, each performed in triplicate (n�3). For western blot analysis,

blots from at least three independent experiments were used for densi-

tometry analysis (n�3). Statistical analysis of the data was performed

using one-way ANOVA followed by Bonferroni test, and statistically

significant values were those with an alpha level of 0.005 or below.

Error bars represent SEM. *P<0.05; **P<0.01; ns, not statistically sig-

nificant (P>0.05).

3 | RESULTS

3.1 | Cytotoxic effects of diquat in PC12 cells

In this study, the role of diquat as a neurotoxicant was examined

using PC12 cells, a widely accepted model for studying neuronal cell

death induced by pesticides. To determine the effects of diquat on

PC12 cells, cellular morphology was assessed (Figure 1A). Exposure

of PC12 cells to media containing PBS had no effect on cell mor-

phology. However, diquat treatment (20 and 40 lM) had a dramatic

effect on the shape of PC12 cells. At these diquat concentrations,

cell density was reduced, and their shape became more rounded. To

further assess diquat-induced cytotoxicity, PC12 cells were treated

with various concentrations (0–40 lM) of diquat for 24 h. Subse-

quently, cell viability and cytotoxicity were measured by MTS and

LDH assays. Consistent with cellular morphology results, diquat

administration significantly decreased cell viability and increased

LDH release in the presence of 5–40 lM of diquat (Figure 1B) in a

concentration-dependent manner. To determine the effects of

diquat-induced neurotoxicity specifically in dopaminergic neurons,

we examined the effect of diquat on DA level in PC12 cells. DA

level was analyzed at 24 h after treatment with diquat. As shown in

Figure 1C, diquat treatment significantly reduced DA level in a

concentration-dependent manner.

3.2 | Involvement of reactive oxygen species

in diquat-induced apoptotic cell death

Enzymes that defend against oxidative stress, such as MnSOD are

present in the neuron. To measure oxidative stress induced by

diquat, PC12 cells were treated with diquat for 24 h. MnSOD levels

were reduced in the diquat-treated cells compared to control cells

(Figure 1D). These results indicate that increased oxidative stress

induced by diquat is associated with altered expression of antioxi-

dant enzymes.

To ascertain whether diquat-induced toxic effects on PC12 cells

are responsible for increased oxidative stress, PC12 cells were exposed

to 20 lM diquat for 4 h, and ROS levels were measured using DCF-

DA, a membrane permeable non-fluorescent dye. The membrane per-

meable dye is cleaved to H2DCF by intracellular esterase, and then the

cleaved dye is oxidized by ROS to produce green fluorescence. We

performed DCF-DA labeling and analysis using a microscope and FACS

system to detect ROS generation by diquat. Diquat increased H2DCF-

DA fluorescence intensity relative to the control, indicating generation

of ROS. The accumulation of ROS was reversed by the simultaneous

presence of 5 mM of antioxidant NAC (Figure 1E, Supporting Informa-

tion Figure S1). In addition, diqaut-induced apoptosis was attenuated

by NAC. Hence, the results suggest that diquat toxicity is caused in

part by generation of ROS.

To study whether oxidative stress is responsible for the cell loss

caused by exposure to diquat, 5 mM of NAC were used as shown in

Figure 1E. ROS accumulation and cell death with diquat exposure were

markedly inhibited in cells pre-treated with NAC. Diquat-induced acti-

vation of caspase-3 following diquat treatment was also prevented by

NAC pretreatment (Figure 1E). These results support the idea that pro-

duction of ROS initiates diquat-induced apoptosis.

3.3 | Diquat leads to mitochondrial dysfunction

via inhibition of mitochondria complex I activity

Disruption of mitochondrial respiratory function is one consequence of

oxidative stress. The lack of mitochondrial complex I activity can affect

dopaminergic neurons, which are more susceptible to oxidative stress,

and is a major source of superoxide anions, suggesting that complex I is

associated with increased mitochondrial ROS production and redox sig-

naling. To determine if mitochondrial complex I activity is inhibited by

diquat in PC12 cells, mitochondrial complex I activity was analyzed

using the same mitochondrial preparation method as in the cytochrome

c release assay. As shown in Figure 1F, diquat inhibited mitochondrial

complex I activity in a concentration-dependent manner, and 40 lM

diquat attenuated complex I activity by about 53% compared to control

cells. To study the ATP level in mitochondria, we used a commercially

available ATP determination kit. Cellular ATP level was measured using

a bioluminescence assay. Exposure to 20-lM diquat for the indicated

times (0, 1, 2, 4 h) reduced the level of ATP compared to control cells

(Figure 1F). Collectively, these results strongly suggest that diquat

exposure induces mitochondrial dysfunction.

4 | CHOI ET AL.

FIGURE 1 Diquat induces mitochondrial dysfunction and involves intrinsic mitochondrial-dependent apoptosis. A, Changes in cellular morphologywere observed using an invertedmicroscope (x 20). B, Cytotoxicity was measured byMTS assay and LDH assay after 24 h (n55). C, Intracellularamounts of DAweremeasured. D,Western blot analysis ofMnSOD in PC12 cells after various concentrations (0–40 lM) exposures to diquat for24 h. E, PC12 cells were labeledwithH2DCF-DA (10 lM) and ROS generationwas examined. Intensity of fluorescencewas quantified by flow cytom-etry. Treatment with 20 lMdiquat increased fluorescence due to ROS generation. ROS accumulation was reversed by adding 5mMNAC. NAC sup-pressed diquat-induced cytotoxicity. NAC suppressed diquat-induced cleavage of caspase-3.Western blot for b-actin was carried out to confirmequal loading of the extracts. Each blot represents three independents experiments performed on PC12 cells. F, Crucial mitochondrial proteins (200lg) were analyzed using themitochondrial complex I enzyme activity microplate assay kit. Intracellular ATP level wasmeasured using a biolumines-cence assay and ATP determination kit. ATP values are expressed as percentage of control. G, Effects of diquat on nuclear morphology in PC12 cells.Changes in nuclear morphology were observed byHoechst 33342 staining. Nuclei of control cells were stained homogeneously andwere less bright,whereas the nuclei of diquat-treated cells appeared hyper-condensed (brightly stained), and showed chromatin fragmentation (arrow). Dense andfragmented nuclei were counted as apoptotic. H,Western blot analysis of cytochrome c, Bax, and Bcl2 with mitochondrial/cytosolic proteins. Allsamples of 30 lg mitochondrial or cytosolic protein were loaded onto 15% SDS-PAGE gels. a-Tubulin was used to confirm equal loading of cytosolicproteins, and COX IVwas used to confirm equal loading of mitochondrial proteins. Error bars show standard error of themean (SE) (n53). *P<0.05,**P<0.01with respect to the control. ##P<0.01with respect to diquat treatment (n53) [Color figure can be viewed at wileyonlinelibrary.com]

CHOI ET AL. | 5

3.4 | Mitochondria-mediated apoptotic characteristics

of diquat-induced neuronal cell death

We investigated nuclear morphology and caspase activation to con-

firm the type of cell death in PC12 cells exposed to diquat. Nuclei

were stained with Hoechst 33342 to assess changes in nuclear

shape and chromatin integrity by fluorescence microscopy (Figure

1G). Nuclei in the control cultures exhibited normal shapes and

uniformly stained chromatin, and cells were round and large. How-

ever, when cells were exposed to 40 lM diquat for 24 h, the

nuclei exhibited condensed and fragmented chromatin, a typical

feature of apoptotic cell death. The number of apoptotic nuclei

increased with diquat exposure by about 30% compared to con-

trols (Figure 1G).

Next, to determine the possible contribution of an apoptotic

caspase-dependent pathway in this study, we tested whether diquat

exposure activated caspase-9 and caspase-3. Exposure to diquat

enhanced the activity of caspase-9 and caspase-3 in a

concentration-dependent manner (Supporting Information Figure S2).

One rate-limiting stage in apoptotic cell death is alteration of mito-

chondrial permeability, accompanied by the release of pro-apoptotic

factors such as cytochrome c. Western blotting from mitochondrial

and cytosolic fractions isolated from control and diquat-treated cells

showed that cytochrome c release from mitochondria produced a

concentration-dependent increase after diquat treatment compared

to the control (Figure 1H). In addition, cell treatment with diquat

increased the protein level of Bax and decreased the protein level

FIGURE 1 Continued

6 | CHOI ET AL.

of Bcl-2 (Figure 1H). These findings suggest that diquat can cause

apoptotic cell death via a mitochondria-mediated intrinsic apoptotic

pathway.

3.5 | Diquat activates NF-jB and p53 pathways

to contributes to diquat-induced cell death

We next investigated whether NF-jB and p53 play a regulatory role in

diquat-induced cell death. Because NF-jB is involved in regulating apo-

ptosis in several systems, we examined the effect of diquat on NF-jB

expression. Treatment with 20 lM diquat significantly increased phos-

phorylation of NF-jB in a time-dependent manner (Figure 2A) but

decreased expression of p-NF-jB at 24 h after treatment. Diquat treat-

ment also greatly decreased the protein level of IjB, an inhibitor pro-

tein of NF-jB (Figure 2A). In addition, translocation of NF-jB from the

cytosol to the nucleus increased in diquat-treated cells compared to

controls (Figure 2B).p53 expression is known to be involved in apopto-

tic signaling in neuronal cell death. Treatment with 20 lM diquat

increased p53 protein level in whole cell lysate in a time-dependent

manner (Figure 2A). As shown in Figure 2B, nuclear accumulation of

p53 increased following diquat treatment, and the level of cytosolic

p53 was also enhanced. Therefore, diquat increased expression of both

NF-jB and p53 in the nuclei of PC12 cells.

3.6 | Inflammatory response involves diquat-induced

apoptosis

To clarify whether the toxic effects induced by diquat on PC12 cells

resulted in increased inflammatory stimulation, we first confirmed the

levels of COX-2 and TNF-a expression in diquat-treated cells. Cells

FIGURE 2 Effects of diquat on NF-jB or p53 expression in cytosolic and nuclear fractions of diquat-treated PC12 cells. A, Western blotanalysis of p-NF-jB, IjB, and p53 in PC12 cells after exposure to 20 lM diquat for indicated times (1–24 h). Levels of p-NF-jB and p53increased with diquat exposure in time-dependent manner. However, level of IjB decreased with diquat exposure. B, Western blot analysisof NF- jB and p53 in cytosolic and nuclear protein extracts. Diquat treatment increased nuclear levels of NF-jB and p53 in aconcentration-dependent manner. Densities of protein bands were quantified using scanning densitometry. NF-jB and Lamin B or p53 and

Lamin B ratios are shown in each panel. Error bars denote standard error of the mean (SE). *P<0.05, **P<0.01 compared to control cells

CHOI ET AL. | 7

were exposed to 20 lM diquat for the indicated times (0 min, 30 min,

1 h, 3 h, 6 h, 12 h, and 24 h). Addition of diquat provoked a rapid

increase in the expression of COX-2 after 30 min of treatment, result-

ing in a maximum at 6 h (Figure 3A). Exposure to diquat significantly

enhanced the expression of COX-2 and TNF-a in a concentration-

dependent manner (Figure 3B). Together, these results suggest that

diquat treatment led to increased expression of inflammatory genes

such as COX-2 and TNF-a, suggesting that an inflammatory response

might be involved in diquat-induced apoptotic signaling in PC12 cells.

3.7 | NF-jB regulates inflammatory responses via p53

signaling

To evaluate the role of the inflammatory response mediated by NF-jB-

dependent p53 activity in diquat toxicity, the effects of SN50 and

pifithrin-a on diquat-induced inflammatory responses were assessed.

SN50 is a cell permeable inhibitor of NF-jB nuclear translocation, and

pifithrin-a is an inhibitor of p53 transactivation. The diquat-induced

inflammatory response was attenuated by pre-treatment with SN50

(Figure 4A). In addition, pifithrin-a pretreatment blocked increases of

COX-2 and TNF-a expression in diquat-treated cells (Figure 4B). Inter-

estingly, pre-treatment with SN50 effectively blocked diquat-induced

phosphorylation of NF-jB and significantly reduced the increase in

p53 expression caused by diquat (Figure 4A,C). SN50 or pifithrin-a

both alter nuclear translocation in diquat-treated cells (Figure 4A,B, left

panel). The results suggest that NF- jB can promote an inflammatory

response in diquat-treated cells to promote cytotoxic insult through

upregulation of p53 signaling. Diquat-induced cytotoxicity was attenu-

ated by pre-treatment with SN50 or pifithrin- a (Figure 4D). In addi-

tion, pretreatment of SN50 and pifithrin- a effectively blocked diquat-

induced cleaved caspase-3 expression (Figure 4D).

3.8 | Meloxicam protects against diquat-induced

mitochondrial dysfunction and apoptosis

To evaluate whether meloxicam rescues mitochondrial dysfunction

in diquat-treated cells, we performed mitochondrial complex I activ-

ity assays. Diquat treatment reduced mitochondrial complex I activ-

ity, and meloxicam pretreatment rescued mitochondrial dysfunction

(Figure 5A). We next characterized the protective effects of meloxi-

cam on diquat-induced cytotoxicity. We performed an MTS and

LDH assay by pretreating cells with 40 lM meloxicam prior to

diquat treatment. As shown in Figure 5B, diquat significantly

decreased cell viability and increased cytotoxicity. Specifically, pre-

treatment of cells with meloxicam reversed the cytotoxic effects of

diquat. Furthermore, to determine the effect of meloxicam on

diquat-induced apoptosis, we performed Western blot analysis to

detect expression of cleaved caspase-9 and cleaved caspase-3 in

cells pretreated with meloxicam. Compared to the control group,

treatment with diquat (20 lM) for 24 h significantly increased both

caspase-9 and caspase-3 activity, whereas pretreatment with meloxi-

cam reversed this effect (Figure 5C). In addition, meloxicam inhibited

COX-2 expression in diquat-treated cells. Finally, we stained cells

with a DNA dye to visualize changes in nuclear morphology and

chromatin integrity induced by diquat. The nuclei of control cells

were stained homogenously and less brightly than those of diquat-

treated cells. Conversely, exposure to diquat resulted in apoptosis as

revealed by the appearance of condensed (bright blue) nuclei in

Hoechst-stained cultures. Consistent with our above observations,

meloxicam significantly decreased nuclear condensation and frag-

mentation (Figure 5D). These results demonstrate that meloxicam

effectively blocked diquat-induced apoptotic cell death through res-

cue of mitochondrial dysfunction.

FIGURE 3 Diquat induces inflammatory responses. A, Western blot analysis of COX-2 in PC12 cells after exposure to 20 lM diquat forindicated times (0.5–24 h). Addition of diquat provoked a rapid increase in expression of COX-2 after 30 min of treatment, reaching a maxi-mum at 6 h. B, After diquat treatment, activation states of COX-2 and TNF-a were measured by western blot. Exposure to diquat signifi-cantly enhanced expression of COX-2 and TNF-a in a concentration-dependent manner. Samples were loaded onto 7.5 and 15% SDS-PAGEgels (30 lg protein per lane). Densities of protein bands were quantified using scanning densitometry. COX-2 and b-actin or TNF-a andb-actin ratios are shown in each panel. Error bars denote standard errors of the mean (SE). *P<0.05, **P<0.01 compared to control cells.Each western blot image is representative of three different experiments

8 | CHOI ET AL.

FIGURE 4 Anti-inflammatory effects of NF-jB or p53 inhibitors on diquat-treated cells. Cells were pre-treated with SN50 or pifithrin-a for 1 hand then incubated with 20 lMdiquat for 6 h. A, Pretreatment of SN50 blocked expression of COX-2 and TNF-a. SN50 has effective on nucleartranslocation in diquat-treated cells (left panel). B, Pretreatment with pifithrin-a blocked expression of COX-2 and TNF-a. Pifithrin-a has effec-tive on nuclear translocation in diquat-treated cells (left panel). C, Expression of p53 decreased after pre-treatment with NF-jB inhibitor SN50 indiquat-treated cells. Lamin B was used to confirm equal loading of nucleus proteins. D, Effects of SN50 and pifithrin-a on diquat-induced apopto-sis. LDH assays for cytotoxicity analysis (n55). Western blot analysis of cleaved caspase-3 levels. Samples containing 30 lg of protein wereloaded in 15% SDS–PAGE gels, and blots were probed with corresponding antibodies. b-actin level was used as a loading control. Error bars showstandard error of the mean (SE) (n53). *P<0.05, **P<0.01 with respect to control. #P<0.05, ##P<0.01 relative to diquat alone

CHOI ET AL. | 9

4 | DISCUSSION

The objective of this study was to determine whether diquat induces

cytotoxicity in PC12 cells used as a PD cellular model and to identify

the underlying molecular mechanisms. Exposure of PC12 cells to

diquat led to ROS production and intrinsic apoptotic cell death via

caspase-9 and caspase-3 activation. In addition, diquat inhibited mito-

chondrial complex I activity, and the resulting excessive ROS genera-

tion triggered cytochrome c release from mitochondria to cytosol in

diquat-treated cells. We also demonstrated that diquat-induced

inflammatory response involved p53 signaling via activation of NF-jB,

and the inflammatory response caused apoptosis. Together, our find-

ings suggest that diquat induces mitochondrial-dependent apoptosis

and its NF-jB activation shows a pro-apoptotic role through p53

signaling.

Diquat was cytotoxic to PC12 cells, and induced decrease of DA

level, indicating that cell death induced by diquat was due to cytotox-

icity of dopaminergic neuronal components in these cells. Incubation

of PC12 cells with diquat increased ROS production, and diquat-

induced cell death was attenuated by pre-treatment of cells with the

antioxidant, NAC. These results indicate that oxidative stress is

involved in diquat-induced neurotoxicity in PC12 cells, consistent

with the ROS-mediated toxicity of pesticides in this cell line. An accu-

mulating body of evidence suggests that oxidative stress is one of

the most important factors leading to neuronal cell death in PD.30

The 1-methyl-4-phenylpyridinium (MPP1) might be involved in neu-

ronal cell death and implies a systemic defect in mitochondrial com-

plex I that causes deleterious effects through oxidative damage.31 In

addition, pesticides such as rotenone and chlorpyrifos caused neuro-

toxicity and mitochondrial dysfunction by inhibiting mitochondrial

complex I activity.10,32 Mitochondrial complex I inhibition and subse-

quent oxidative stress are thought to be central to dopaminergic cell

death.33 Mitochondrial dysfunction also induces cytochrome c release

from mitochondria and results in apoptosis through caspase-3 activa-

tion. To assess the validity of this notion, we performed mitochondrial

complex I activity and cytochrome c release assays to confirm that

diquat-induced mitochondrial dysfunction is followed by ROS genera-

tion, cytochrome c release, and caspase-3 activation. As shown in Fig-

ure 1F, diquat inhibited mitochondrial complex I activity in PC12 cells

compared to the control. This inhibition led to decreased ATP level.

These results implicate diquat-induced apoptosis in the inhibition of

mitochondrial complex I activity. These results are the first evidence

of a mitochondria-dependent cytotoxic effect of diquat in a culture

system. To define the characteristic features of apoptosis in diquat-

treated PC12 cells, we assessed the intrinsic mitochondrial apoptotic

pathway based on release of cytochrome c and activation of

caspases-9 and caspase-3. Cytochrome c was released from mito-

chondria into the cytosol after diquat treatment. We also detected

caspase-9 and caspase-3 activation, and DNA damage in diquat-

treated cells. These results suggest that diquat-induced apoptotic cell

death is mediated by ROS generation linked to the intrinsic mitochon-

drial apoptotic pathway.

One transcription factor activated by oxidative stress is NF-jB.

Once activated, NF-jB increases the expression of many genes

involved in promoting cell death or survival.34 In the present study,

diquat resulted in increased level of p-NF-jB in PC12 cells and

induced translocation of NF-jB from cytosol into nuclei in a

concentration-dependent manner. Diquat also decreased protein lev-

els of IjB, a member of the family of proteins that inhibit NF-jB.

SN-50, an inhibitor of NF-jB nuclear translocation, also attenuated

diquat-induced cell death. The proposed involvement of NF-jB is

consistent with in vitro studies of pesticides such as paraquat and

MPP1 ion.35,36 In a previous study, we reported the pro-apoptotic

role of NF-jB in chorpyrifos-treated human neural precursor cells.37

Among potential candidates for this role, p53 is of particular inter-

est. Here, we found that diquat increases the expression level of

p53. Induction of this gene is closely related to NF-jB activation by

IjB degradation and subsequent apoptosis. In addition to its well-

known function as a transcription factor, p53 induces apoptosis.21,38

Diquat altered the level of p53 protein with concomitant accumula-

tion of nuclear p53 in these cells, consistent with reports of the

effects of other pesticides on neuronal cells.39,40 In addition, pre-

treatment with pifithrin-a, a specific p53 inhibitor, decreased diquat-

induced cell death. Pifithrin-a can selectively inhibit p53 transcrip-

tional activity in cell lines and prevent DNA damage.41 The possibil-

ity that NF-jB activation triggers p53 induction and apoptotic cell

death in PC12 cells was addressed by studies with SN50. We found

that SN50 attenuated the diquat-induced increase in expression of

FIGURE 4 Continued

10 | CHOI ET AL.

both NF-jB and p53 protein. These results suggest that NF-jB

upregulates expression of p53 in PC12 cells. Our data from a PC12

cell model of neuroblast cell death induced by diquat implicate

involvement of a signaling network in which NF-jB and p53 coordi-

nate to activate the conserved mitochondrial pathway of death

involving Bax activation, cytochrome c release, and caspase

FIGURE 5 Meloxicam rescues diquat-induced mitochondrial dysfunction and apoptosis. A, Crucial mitochondrial proteins (200 lg) were ana-lyzed using the mitochondrial complex I enzyme activity microplate assay kit. Mitochondrial complex I enzyme activity is expressed as a percent-age of the control. B, Cell viability and cytotoxicity was measured byMTS assay and LDH assay (n53). C, Western blot analysis of COX-2,cleaved caspase-9, and cleaved caspase-3 levels. Samples containing 30 lg of protein were loaded in 7.5, 10, and 15% (COX-2, cleaved caspase-9and cleaved caspase-3) SDS–PAGE gels, and blots were probed with corresponding antibodies. b-actin level was used as a loading control. D,Effects of diquat on nuclear morphology of PC12 cells. Changes in nuclear morphology were observed by Hoechst 33342 staining and fluores-cence microscopy. Fragmentation of nuclei into oligonucleosomes and chromatin condensation were noted. Dense and fragmented nuclei werecounted as apoptotic cells. Error bars represent standard error of the mean (SE). *P<0.05, **P<0.01 with respect to control conditions (DMSO);# P<0.05, ##P<0.01 with respect to diquat treatment [Color figure can be viewed at wileyonlinelibrary.com]

CHOI ET AL. | 11

activation. Diquat altered expression levels of NF-jB and p53 pro-

teins, which are involved in cell death signaling. This resulted in

accumulation of both nuclear and cytosolic NF-jB and p53 in these

cells. Activated NF-jB can potentially regulate p53-mediated proc-

esses; for example, NF-jB can directly regulate expression of p53

via NF-jB sites in the promoter sequence of the p53 gene.42–44

The role of NF-jB in inflammation was anticipated from the early

phase of its discovery; it is activated by various cytokines to subse-

quently activate the same and other proinflammatory cytokines, che-

mokines, adhesion molecules, acute phage proteins, inducible

effector enzymes, and regulators of cell proliferation and apopto-

sis.21 We found that NF-jB inhibitor attenuated inflammatory

responses in diquat-treated cells.

Inflammation is involved in neuronal death and a wide range of

proinflammatory factors such as TNF-a, IL-1b, and IL-6 might be toxic

to neurons.45,46 In addition, there is convincing evidence for the

involvement of inflammation caused by increased expression of COX-

2.47,48 It was recently demonstrated that environmental toxicants

including paraquat and rotenone exert their toxicity through a pathway

involving COX-2 in dopaminergic neurons.36,49,50 Therefore, based on

these reports, we noted that exposure of PC12 cells to diquat resulted

in elevated expression of COX-2 and subsequently increased produc-

tion of pro-inflammatory cytokines such as TNF-a. Both COX-2 and

TNF-a were upregulated by diquat treatment in these cells, and the

expression of these proteins was attenuated by cell pretreatment with

the COX-2 inhibitor, meloxicam. Together, these results suggest that

oxidative stress is a main cytotoxic pathway in diquat-treated cells. Pre-

vious studies have reported a correlation between expression of COX-

2 and p53,37,51 and COX-2 has been shown to regulate the transcrip-

tional activity of p53.52 To further investigate the relationship between

p53 and inflammation in diquat-treated cells, we pretreated PC12 cells

with pifithrin-a. Pretreatment with pifithrin-a decreased COX-2

expression in diquat-treated cells. p53 activity has been linked to toll

like receptor (TLR) signaling in diverse mammalian cell types.53,54 TLR is

implicated in innate immunity and neuroinflammatory processes within

the CNS.55 Microglial exposed to LPS showed enhanced expression of

iNOS and enhanced secretion of TNF-a and IL-1b, all of which was

prevented by co-treatment with pifithrin-a.56 In addition, we found

that meloxicam inhibited activation of caspase-9 and caspase-3. Our

results suggest a possible interaction between COX-2 and p53 expres-

sion in diquat-treated cells.

Overall, our findings indicate that diquat induces apoptosis in

PC12 cells through activation of the mitochondrial-dependent apopto-

sis pathway via ROS-dependent activation of p53. Diquat-induced p53

activation can be mediated by activation of the NF-jB pathway. The

inflammatory response associated with diquat-induced apoptosis

involved activation of p53-dependent pathways and ultimately

caspase-3. Among many apoptotic pathways, NF-jB/p53 plays a cru-

cial role in diquat-induced cell death in these cell lines. Finally, these

data suggest that diquat can affect PC12 cells in a similar manner to

several known and suspected neurotoxicants. Diquat-mediated apopto-

sis can be blocked by NF-jB inhibitors, which might have potential clin-

ical relevance.

ORCID

Hyun Chul Koh http://orcid.org/0000-0002-7103-9549

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SUPPORTING INFORMATION

Additional Supporting Information may be found online in the sup-

porting information tab for this article.

How to cite this article: Choi SE, Park YS, Koh HC. NF-jB/p53-

activated inflammatory response involves in diquat-induced

mitochondrial dysfunction and apoptosis. Environmental Toxicol-

ogy. 2018;00:1–14. https://doi.org/10.1002/tox.22552

14 | CHOI ET AL.

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