Toxicology in Vitro 67 (2020) 104883

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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Effects of antioxidants on oxidative stress and inflammatory responses of T

human bronchial epithelial cells exposed to particulate matter and cigarette
smoke extract
Eun Suk Sona,1, Jeong-Woong Parka,1, Yu Jin Kima, Sung Hwan Jeonga, Jeong Hee Hongb,
⁎ ⁎⁎
Se-Hee Kimc, , Sun Young Kyunga,
a
Department of Medicine, Gachon University Gil Medical Center, Incheon, Republic of Korea
b
Department of Physiology, Graduate School of Medicine, Gachon University, Incheon, Republic of Korea
c
Gachon Medical Research Institute, Gachon University Gil Medical Center, Incheon 21565, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Particulate matter (PM) is a type of air pollutant that induces adverse health effects, including acute exacer-
Cigarette smoke extract bation of chronic obstructive pulmonary disease (COPD). However, the effects of co-exposure to PM and ci-
COPD garette smoke extract (CSE) on bronchial epithelial cells remain unknown. This study investigated the cytotoxic
Inflammation and pro-inflammatory effects of combined exposure to PM and CSE on bronchial epithelial cells, and assessed the
Particulate matter
potential of antioxidants to inhibit CSE/PM-induced oxidative stress and inflammation. Exposure of epithelial
Sulforaphane
cells to PM or CSE induced cytotoxicity, inflammation, and oxidative stress, all of which were dramatically
Sulforaphane N-acetyl-L-cysteine
increased when cells were exposed to the combination of CSE and PM. Importantly, the adverse effects of CSE/
PM exposure were suppressed when cells were treated with sulforaphane (SFN) or sulforaphane N-acetylcysteine
(SFNAC). Furthermore, SFN and SFNAC suppressed the CSE/PM-induced pro-inflammatory cytokine production
and expression of inflammatory genes. Combined PM and CSE exposure further activated the MAPK and Nrf2
signaling pathways. SFN and SFNAC attenuated CSE/PM-induced epithelial toxicity through the ERK/JNK sig-
naling pathway-dependent inhibition of inflammation. Moreover, SFN and SFNAC suppressed ROS generation by
activating antioxidant enzymes and Nrf2 signaling. Therefore, SFN and SFNAC could be a promising approach to
prevent or mitigate the exacerbation of pulmonary diseases caused by PM and other air pollutants.

1. Introduction
Air pollution and particulate matter (PM) have become a serious
health problem worldwide, with 3 million estimated premature PM-
related deaths per year (Organization, 2016). PM is a mass concentra-
tion of particles less than 10 μm (PM10) or less than 2.5 μm (PM2.5).
These particles can penetrate the respiratory tract from the nasal pas-
sages to the alveoli in the lungs due to their high penetrability (Gilmour
et al., 2007). Patients with chronic pulmonary diseases are more sus-
ceptible to PM; exposure to PM in these patients increases the in-
cidence, exacerbation, and mortality of pulmonary conditions (Pope III
et al., 2002; Pope III and Dockery, 2006). Several studies have ad-
dressed the respiratory hazards of PM, as the respiratory system is the
primary entry site (Gan et al., 2013; Pope III and Dockery, 2006;
Thurston et al., 2016). Increased exposure to PM is associated with
mortality and risk for lung cancer (Pope III et al., 2002).
Acute exacerbation of chronic obstructive pulmonary disease
(COPD) poses several challenges in healthcare, as it has long-term
detrimental effects on patients, accelerates disease progression, and
increases healthcare costs significantly. Viral infections are important
causes of COPD and patient hospitalization due to disease exacerbation.
Recent in vitro and epidemiological studies have reported that cigarette
smoke (CS) and PM are important factors contributing to acute ex-
acerbation of COPD (Krimmer and Oliver, 2011). In addition, fine PM
has been found to enhance cigarette smoke extract (CSE)-induced in-
flammation and apoptosis in COPD models and bronchial epithelium

⁎
Correspondence to: S-H. Kim, Gachon Medical Research Institute, Gachon University Gil Medical Center, Incheon 21565, Republic of Korea.
⁎⁎
Correspondence to: S. Y. Kyung, Department of Medicine, Gachon University Gil Medical Center, 21 Namdong-daero 774 beon-gil, Namdong-gu, Incheon,
Republic of Korea.
E-mail addresses: sehee0423@gilhospital.com (S.-H. Kim), light@gilhospital.com (S.Y. Kyung).
1
Contributed equally

https://doi.org/10.1016/j.tiv.2020.104883
Received 3 January 2020; Received in revised form 1 May 2020; Accepted 4 May 2020
Available online 06 May 2020
0887-2333/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

A
120

100

Cell viability (% of control)

*
80
**
60

40

20

0
Control 1 5 10 20
CSE (%, v/v)

B ** C **
*** *** 2.5 *** **
120

LDH release ratio ( of control)

Cell viability (% of control)

100
2
80

60 1.5

40
1
20

0 0.5
Control PM10 CSE CSE+PM10 Control PM10 CSE CSE+PM10

Fig. 1. Effects of CSE and PM10 on the viability of BEAS-2B epithelial cells. (A) Cells were treated with different concentrations of CSE for 7 days, and cell viability
was assessed using the MTT assay. (BeC) Cells were treated with 1% CSE for 7 days, followed by exposure to 100–200 μg/mL PM10 for 24 h. Cell viability and
cytotoxicity were determined by MTT and lactate dehydrogenase (LDH) assays, respectively. All experiments were independently performed three times. Data are
expressed as mean ± standard deviation (SD) (n = 5). *P < .05, **P < .005 compared to the control group. CSE, cigarette smoke extract; PM, particulate matter.

(Wang et al., 2019). Based on these findings, we believe that PM ag- expression. As the role of PM exposure and subsequent inflammation in
gravates CSE-induced oxidative stress and inflammation in bronchial CSE-induced COPD exacerbation remains unclear, in this study, we
epithelial cells and that antioxidants could protect from the adverse evaluated the effects of PM on CSE-induced bronchial injury. We also
effects of CSE and PM via the Nrf2 and MAPK pathways. used the antioxidants SFN and SFNAC to assess the role of reactive
The pathophysiology of PM-induced cytotoxicity is characterized by oxygen species (ROS) in CSE/PM-induced bronchial cell injury, as well
oxidative stress-mediated inflammatory responses (González-Flecha, as explore their potential use to reduce inflammatory responses.
2004; Mazzoli-Rocha et al., 2010; Øvrevik et al., 2015; Valavanidis
et al., 2013). When PM penetrates the lower respiratory tract, it initially
2. Materials and methods
affects alveolar macrophages and epithelial cells. Long-term exposure
to PM can lead to inflammation and apoptosis due to the activation of
2.1. Cell culture and chemicals
epithelial cells and macrophages and the subsequent release of med-
iators in response to oxidative stress. Therefore, oxidative stress and
Human lung epithelial cells (BEAS-2B) were obtained from Lonza
inflammation are essential for COPD initiation and progression. How-
Biologics (Wokingham, UK). The cells were maintained in Dulbecco's
ever, there has been no report suggesting that CSE-induced COPD ex-
modified Eagle's medium (DMEM) (Welgene, Daegu, Korea) supple-
acerbates through inflammation aggravation by PM exposure. As such,
mented with 10% fetal bovine serum (Welgene), 1% penicillin, and 1%
we aim to evaluate the effects of PM on CSE-induced bronchial injury
streptomycin (Welgene). SFN and SFNAC were purchased from Cayman
and the protective effects of antioxidants on adverse effects of PM and
Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide
CSE in cell culture system.
(DMSO).
Sulforaphane (SFN) is an organic isothiocyanate found in cruci-
ferous vegetables, such as broccoli (Hanlon et al., 2009). Many studies
have reported that SFN has anti-apoptotic, anti-inflammatory, anti- 2.2. Exposure to cigarette smoke extract (CSE), particulate matter (PM),
oxidant, and cytoprotective effects when used at low concentrations. and antioxidants
Furthermore, sulforaphane N-acetylcysteine (SFNAC), a primary meta-
bolite of SFN, induces the expression of various chemo-preventive en- The reference cigarettes (3R4F) were purchased from the Tobacco
zymes via Keap1-Nrf2 signaling and ARE-driven regulation of gene Research Institute (University of Kentucky, Lexington, KY, USA). The
CSE was prepared as per a previously described method (Son et al.,

2
E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

A 450 Control+PM B 2500 Control+PM ***

400 CSE+PM *** CSE+PM
*
*** 2000
350

IL-6 cytokine (pg/mL)

IL-8 cytokine (pg/mL)

300 ***
1500
250
***
200
1000
150
100
500
50
0 0
0 100 200 0 100 200
PM10 (μg/mL) PM10 (μg/mL)

C DC FH - DA Bright field DCFH-DA Bright field

Control CSE

PM10
CSE+PM10

NS

D *
4 NS *
Relative DCFH-DA intensity

NS
3

0
Control PM10 CSE CSE+PM10

Fig. 2. Effects of PM10 on CSE-induced inflammation and oxidative stress in BEAS-2B epithelial cells. Cells were treated with 1% CSE for 7 days, followed by exposure
to 100–200 μg/mL PM10 for 24 h. (A, B) IL-6 and IL-8 levels were measured in cell supernatants. (C) The intracellular ROS levels were measured using the fluorescent
probe DCFH-DA. (D) Quantitative analyses of fluorescence intensity for DCFH-DA. All experiments were independently performed three times. *P < .05,
**P < .005, ***P < .0005 compared to control groups. CSE, cigarette smoke extract; PM, particulate matter. Magnification, ×400.

2018). The standard reference PM (PM10, SRM 1648) was obtained 2.3. Cytotoxicity and viability assays
from the National Institute of Standards and Technology (NIST, Gai-
thersburg, MD, USA). PM10 was suspended in Dulbecco's phosphate- Lactate dehydrogenase (LDH) release was measured using a cyto-
buffered saline (DPBS) and diluted to appropriate concentrations. PM10 toxicity detection kit (Takara Bio, Dalian, China) according to the
suspension was added to the cells immediately after vortexing. manufacturer's protocol. Cell viability was measured with 3-[4,5-di-
BEAS-2B cells were seeded in 100 mm dishes and incubated in methylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Cells
growth medium with or without 1% CSE for 7 days; the growth medium were exposed to CSE and/or PM, and MTT reagent was added to each
was refreshed every 2–3 days. After 7 days, control cells and the CSE- well. After 2 h incubation, the formazan crystals were dissolved in
exposed cells were seeded at densities of 1 × 105 cells/mL in dishes DMSO. Absorbance was measured at 562 nm. All experiments were
containing the appropriate cell culture medium. After 24 h, independently performed three times.
100–200 μg/mL PM10 and/or 10 μM SFN or 10 μM SFNAC were added
to the cells, followed by incubation for an additional 24 h.

3
E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

A
Control CSE/PM10 CSE/PM10+SFN CSE/PM10+SFNAC

DCFH-DA

MitoSox

TMRE

B
** ** ** * * *
** * ***
140 7
350
120 6
300
MitoSox intensity
DCFH-DA intensity

TMRE intensity

100 5
250
80 4
200
60 3
150
100 40 2

50 20 1

0 0 0

Fig. 3. Effects of antioxidants on CSE/PM10-induced ROS generation and oxidative stress in BEAS-2B epithelial cells. Cells were treated with 1% CSE for 7 days,
followed by a 24 h exposure to 100 μg/mL PM10 in combination with 10 μM SFN or 10 μM SFNAC. (A) Detection of ROS levels and mitochondrial dysfunction. Cells
were stained with DCFH-DA, Mitosox, and TMRE. Magnification, ×400. (B) Quantitative analyses of DCFH-DA, Mitosox, and TMRE fluorescence intensities. (C-E)
NADPH oxidation, malondialdehyde (MDA) concentration, and Glutathione (GSH) level in BEAS-2B cells treated with CSE/PM10 with or without 10 μM SFN or 10 μM
SFNAC for 24 h. All experiments were independently performed three times. *P < .05, **P < .005, ***P < .0005 compared to control groups. NS, not significant;
CSE, cigarette smoke extract; PM, particulate matter; SFN, sulforaphane; SFNAC, sulforaphane N-acetylcysteine.

4
E.S. Son, et al.
C ** NS D ***
* *
9 0.6
MDA activity (μmol/mg prot)
NADP/NADPH ratio
6 0.4
3 0.2
0 0.0
Fig. 3. (continued)
2.4. Assessment of oxidative damage
The levels of glutathione (GSH, Cell Biolabs Inc., San Diego, CA,
USA) and malondialdehyde (MDA, Cell Biolabs Inc.) were measured as
per the instructions of each assay kit. Total GSH level and MDA content
were measured at 405 nm and 550 nm, respectively. All experiments
were independently performed three times.
2.5. Nuclear factor E2-related factor (Nrf2) transcriptional activity
The transcriptional activity of Nrf2 was measured using the Nrf2
Transcription Factor Assay Kit (Abcam, Cambridge, MA, USA) ac-
cording to the manufacturer's instructions. The nuclear proteins were
incubated with antioxidant response element (ARE) consensus oligo-
nucleotides (5’-GTCACAGTACTCAGCAGAATCTG-3′), and Nrf2-ARE
binding was assessed using an anti- Nrf2 antibody and appropriate
HRP-conjugated secondary antibody. The specific activity of the tran-
scription factor was determined at 450 nm. All experiments were in-
dependently performed three times.
2.6. Western blotting analyses
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer
(150 mM NaCl, 50 mM Tris-HCl at pH 8.0, 0.1% sodium dodecyl sulfate
(SDS), 0.5% sodium deoxycholate, 1% NP-40, protease inhibitors).
Primary antibodies for anti-p38, anti-phospho-p38, anti-c-Jun N-term-
inal kinase (JNK), anti-phospho-JNK, anti-extracellular signal-regulated
kinase (ERK), anti-phospho-ERK, and anti-glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) were purchased from Cell Signaling (Beverly,
MA, USA). Band intensities were normalized to GAPDH using the NIH
ImageJ software. Experiments were independently performed three
times.
2.7. Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using the RNAiso Plus reagent (Takara Bio,
Inc., Shiga, Japan), and cDNA was synthesized from total RNA using the
Prime Script RT reagent kit (Takara Bio). qRT-PCR reactions were
prepared using the SYBR Premix kit (Takara Bio) and run on a CFX96
detection system (BioRad, Hercules, CA, USA). Gapdh was used as an
internal control, and all experiments were performed in triplicate. The
primers used for qRT-PCR were as follows: interleukin (IL)-1β-F, 5′-
CCTGCGTGTTGAAAGATGATAA-3′; IL-1β-R, 5′-CTGCTTGAGAGGTGC
TGATGTA-3′; IL-6-F, 5′-GGAAGGTTCAGGTTGTTTTCTGC-3′; IL-6-R,
5′-AAATTCGGTACATCCTCGACGG-3′; IL-8-F, 5′- AAATTTGGGGTGGA
5
Toxicology in Vitro 67 (2020) 104883
*
E * *
**
25
GSH content (μmol/g prot)
20
15
10
5
0
AAGGTT -3′; IL-8-R, 5′-AAGAAACCACCGGAAGGAAC-3′; tumor ne-
crosis factor (TNF)-α-F, 5′-TAGCCCATGTTGTAGCAAACC-3′; TNF-α-R,
5′-ATGAGGTACAGGCCCTCTGAT-3′; chemokine (C-S-C motif) ligand
(CXCL)-F, 5′-CCTCCCTTCTGGTCAGTTG-3′; CXCL-R, 5′-AACCGAAGTC
ATAGCCACAC-3′; monocyte chemoattractant protein (MCP)-1-F,
5′-TGTCCCAAAGAAGCTGTGATC-3′; MCP-1-R, 5′-ATTCTTGGGTTGTG
GAGTGAG-3′; heme oxygenase (HO)-1-F, 5′-CTTCTTCACCTTCCCCAA
CAT-3′; HO-1-R, 5′-TTCTATCACCCTCTGCCTGACT-3′; NAD(P)H dehy-
drogenase (quinone) (NQO)1-F, 5′-AAGCCGCAGACCTTGTGATA-3′;
NQO1-R, 5′- GCAGCGTAAGTGTAAGCAAACT-3′; thioredoxin (TXN)-F,
5′-CATTTCCATCGGTCCTTACAG-3; TXN-R, 5′-ACCCACCTTTTGTCCCT
TCTTA-3′; thioredoxin reductase (TXNRD)-1-F, 5′-CTCTTGGATAGGA
GTTGGTGAA-3′; TXNRD-1-R, 5′-ATGGGCTTGAGACTGGTGACT-3;
GAPDH-F, 5′- TTGGTATCGTGGAAGGACTCA-3′; GAPDH-R, 5′- TGTCA
TCATATTTGGCAGGTTT-3.’
2.8. Interleukin secretion
The levels of secreted IL-6 and IL-8 were measured using cell culture
supernatants. Quantification was performed using the respective ELISA
kits (R&D Systems, Minneapolis, MN, USA) according to the manufac-
turer's instructions. All experiments were independently performed
three times.
2.9. Fluorescence imaging by confocal microscopy
Cellular reactive oxygen species (ROS) generation, mitochondrial
membrane potential (△ψm), and mitochondrial ROS production were
measured using DCFH-DA reagent, TMRE, and MitoSOX Red, respec-
tively. Fluorescent images were acquired using an LSM710 confocal
microscope (Carl Zeiss, Jena, Germany). All experiments were in-
dependently performed three times.
2.10. Statistical analyses
Data are expressed as mean ± standard deviation (SD). One-way
analysis of variance (ANOVA) and Tukey's test were used to compare
data among different groups. P-values less than 0.05 were considered
statistically significant.
3. Results
3.1. Effects of CSE and PM10 on BEAS-2B cell viability
We hypothesized that PM10 might exacerbate cellular toxicity and
E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

** * ** *
700 ** B
A **
2500
IL-6 cytokine (pg/mL) 600

IL-8 cytokine (pg/mL)

2000
500

400 1500

300
1000
200
500
100

0 0

C ** * D *** ***
E *** ***
12 4 35
* *** ***
IL-1β/GAPDH mRNA levels

IL-6/GAPDH mRNA levels

IL-8/GAPDH mRNA levels

10 30
3
25
8
20
6 2
15
4
1 10
2 5

0 0 0

F G H **
*** ** * ** ***
8 1.8 12 *
** **
CXCL-1/GAPDH mRNA levels
MCP-1/GAPDH mRNA levels
TNF-α/GAPDH mRNA levels

1.5 10
6
1.2 8

4 0.9 6

0.6 4
2
0.3 2

0 0 0

Fig. 4. Effects of antioxidants on CSE/PM10-induced inflammation in BEAS-2B epithelial cells. Cells were treated with 1% CSE for 7 days, followed by a 24 h
treatment with 100 μg/mL PM10 in combination with 10 μM SFN or 10 μM SFNAC. (A, B) IL-6 and IL-8 levels in cell supernatants were determined by ELISA. (CeH)
The mRNA levels of IL-1β, IL-6, IL-8, TNF-α, MCP-1, and CXCL-1 were determined by real-time PCR. All experiments were independently performed three times.
*P < .05, **P < .005, ***P < .0005 compared to control groups. CSE, cigarette smoke extract; PM, particulate matter; SFN, sulforaphane; SFNAC, sulforaphane
N-acetylcysteine.

inflammation of chronically CSE-exposed bronchial epithelial cells. To of CSE significantly reduced viability in BEAS-2B cells (Fig. 1A). Based
select the concentration of CSE that does not induce cytotoxicity, we on these results, a non-toxic dose (1%) of CSE was chosen as the optimal
exposed BEAS-2B cells to various concentrations of CSE (1, 5, 10, and concentration for prolonged exposure. In addition, we assessed the ef-
20%, v/v) for 7 days. CSE did not affect cell viability at low con- fects of PM10 on cell viability and cell death in CSE-exposed cells using
centrations (1 and 5%); however, higher concentrations (10 and 20%) the LDH and MTT assays, respectively. PM10 significantly decreased cell

6
E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

* * Fig. 5. Effects of antioxidants on Nrf2 signaling in

A CSE/PM10-exposed BEAS-2B epithelial cells. Cells
5
* were treated with 1% CSE for 7 days, followed by a

Nrf2 transcriptional activity

24 h exposure to 100 μg/mL PM10 in combination
4 with 10 μM SFN or 10 μM SFNAC. (A) Nuclear ex-
tracts were analyzed for Nrf-2 transcriptional ac-
3 tivity. (B-E) The mRNA levels of HO-1, NQO-1, TXN,
and TXNRD-1 were determined by real-time PCR.
2 All experiments were independently performed
three times. *P < .05, **P < .005, ***P < .0005
compared to control groups. NS, not significant;
1 CSE, cigarette smoke extract; PM, particulate
matter; SFN, sulforaphane; SFNAC, sulforaphane N-
0 acetylcysteine.

*** *** **
B C **
**
18 *** 6
NQO-1/GAPDH mRNA levels
HO-1/GAPDH mRNA levels

15 5

12 4

9 3

6 2

3 1

0 0

D *** **
E NS ***
2 * 4 ***
TXNRD-1/GAPDH mRNA levels
TXN/GAPDH mRNA levels

1.5 3

1 2

0.5 1

0 0

viability (63.94%) and increased LDH release (2.14-fold) in CSE-ex- 3.2. PM10 aggravates CSE-induced inflammation and oxidative stress in
posed epithelial cells (Fig. 1B and C). Given that PM10 induced cellular BEAS-2B cells
toxicity in CSE-exposed BEAS-2B cells, we performed apoptosis assay to
examine whether PM10 induces necrotic cell death in CSE-exposed To evaluate the toxicity of PM10 in CSE-exposed epithelial cells, we
BEAS-2B cells (Fig. S1). PM10 did not increase apoptosis in CSE-exposed evaluated the levels of pro-inflammatory cytokines in BEAS-2B cells
cells compared to control cells. These findings suggest that PM10 in- after CSE and/or PM10 exposure by ELISA. Exposure of BEAS-2B cells to
duces cytotoxicity in CSE-exposed BEAS-2B cells, which is not mediated 1% CSE for 7 days resulted in increased secretion of the pro-in-
by apoptotic cell death. flammatory cytokines IL-6 and IL-8 compared to control cells. The
production of IL-6 and IL-8 was further increased when BEAS-2B cells
were treated with PM10 for 24 h after exposure to 1% CSE (Fig. 2A and
B). However, the effects of PM10 on the secretion of pro-inflammatory
cytokines was not dose-dependent; CSE-exposed BEAS-2B cells treated

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E.S. Son, et al. Toxicology in Vitro 67 (2020) 104883

A CSE 1%

- - - SFN SFNAC
0 0 100 100 100 PM10 (μg/mL)

p-ERK

ERK

p-JNK

JNK

p-p38

p38

GAPDH

B *** **
C *** **
D
1.4 * 1.4 * 1.4

1.2 1.2 1.2

p-JNK/Total JNK
p-ERK/Total ERK

p-p38/Total p38
1 1 1

0.8 0.8 0.8

0.6 0.6 0.6
0.4 0.4 0.4
0.2 0.2 0.2
0 0 0

Fig. 6. Effects of antioxidants on signaling pathways in CSE/PM10-exposed BEAS-2B cells. Cells were treated with 1% CSE for 7 days, followed by a 24 h treatment
with 100 μg/mL PM10 in combination with 10 μM SFN or 10 μM SFNAC. (A) Levels of phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-p38, and p38 were
detected by Western blotting. (B-D) Quantitative analyses of phospho-ERK1/2, phospho-JNK, and phospho-p38 levels. All experiments were independently per-
formed three times. CSE, cigarette smoke extract; PM, particulate matter; SFN, sulforaphane; SFNAC, sulforaphane N-acetylcysteine.

with 100 and 200 μg/mL PM10 exhibited a 3.1 and 2.5-fold increase in
IL-8 secretion, respectively, compared to CSE-alone cells. To gain fur-
ther insight into the cytotoxic effects of PM10, we investigated the in-
tracellular ROS levels by measuring DCF fluorescence. Intracellular
ROS production was profoundly enhanced in CSE-exposed cells after
PM10 treatment (Fig. 2C). Collectively, these results suggest that PM10
aggravates CSE-induced oxidative stress and inflammation in bronchial
epithelial cells.
3.3. SFN and SFNAC attenuate ROS generation and oxidative stress
induced by CSE and PM10
To explore the ability of the antioxidants SFN and SFNAC to protect
against oxidative stress in CSE and PM10-exposed cells, we examined
the intracellular ROS and mitochondrial ROS (mtROS) levels. DCF and
MitoSOX fluorescence were elevated in BEAS-2B cells after treatment
with CSE and PM10, while SFN and SFNAC decreased DCF and MitoSOX
fluorescence in CSE and PM10-exposed cells. In addition, we determined
the mitochondrial membrane potential (△ψm) by staining cells with
TMRE. PM10 treatment in CSE-exposed cells resulted in mitochondrial
depolarization and mitochondrial dysfunction, whereas the mitochon-
drial membrane potential recovered after treatment with SFN or SFNAC
(Fig. 3A and B). These results suggest that SFN and SFNAC attenuate
CSE- and PM10-induced ROS generation in bronchial epithelial cells.
We also investigated whether SFN and SFNAC affect oxidative stress
caused by CSE and PM10 treatment. NADPH oxidation and MDA activity
were increased in CSE/PM10-exposed cells, while SFN or SFNAC re-
pressed the increase in NADPH oxidation and MDA activity (Fig. 3C and
D). These findings suggest that SFN and SFNAC repress CSE/PM10-in-
duced oxidative damage in BEAS-2B cells. We also evaluated the levels
of the antioxidant indictor GSH to assess the defense mechanisms
against free radicals. The GSH content in CSE- and PM10-treated BEAS-
2B cells were lower compared to control cells. Although GSH content
increased after SFN treatment, SFNAC treatment did not have profound

8
E.S. Son, et al.
effects on GSH content (Fig. 3D), suggesting that SFN (more than
SFNAC) enhances defense mechanisms against CSE- and PM10-induced
oxidative stress in BEAS-2B cells.
3.4. SFN and SFNAC suppress cytokine secretion and expression of
inflammatory genes induced by CSE and PM10
To investigate the impact of antioxidants on bronchial inflammation
induced by CSE and PM10, we measured the secretion levels of the pro-
inflammatory cytokines IL-6 and IL-8 by ELISA. CSE and PM10 exposure
in BEAS-2B cells increased IL-6 and IL-8 levels in cell culture super-
natants, whereas treatment with SFN or SFNAC reduced IL-6 and IL-8
secretions; SFN was more potent than SFNAC in suppressing IL-6 and IL-
8 production (Fig. 4A and B). As SFNAC was inferior to SFN in sup-
pressing IL-6 and IL-8 secretion in CSE/PM10 treated cells, we assessed
whether SFNAC treatment affects apoptosis. We found that SFNAC did
not affect apoptotic cell death or cellular viability compared to control
or CSE/PM10-treated cells (Fig. S1 and S2). To further explore the ef-
fects of SFN and SFNAC on CSE and PM10-exposed cells at the molecular
level, the mRNA levels of chemokines, adhesion molecules, and in-
flammatory cytokines were investigated using qRT-PCR. SFN and
SFNAC significantly suppressed the upregulation of inflammation-re-
lated genes, IL-6, IL-8, IL-1β, MCP-1, TNF-α, and CXCL-1 after CSE and
PM10 treatment (Fig. 4C-H). The finding that SFN and SFNAC sup-
pressed the expression of inflammation-related genes expression is in
line with their inhibitory effects on the secretion of pro-inflammatory
cytokines in CSE and PM10-exposed cells.
3.5. SFN and SFNAC enhance Nrf2 signaling and its downstream genes
To assess whether the antioxidants SFN and SFNAC play a role in
cellular defense against oxidative stress, we examined whether Nrf2
transcriptional activity and the expression of its key downstream genes
are regulated by SFN or SFNAC in CSE/PM10-exposed cells. Nrf2 tran-
scriptional activity was significantly increased (2.59-fold) in the nuclear
extracts from CSE/PM10-exposed cells compared to control cells; SFN
and SFNAC further enhanced the nuclear transcriptional activity of Nrf2
(Fig. 5A). We also assessed the effects of SFN and SFNAC on the mRNA
levels of several Nrf2 downstream genes, including NQO1, HO-1, TXN,
and TXNRD1 in CSE/PM10-exposed cells using qRT-PCR. SFN and
SFNAC increased the expression of HO-1, NQO1, TXN, and TXNRD1
(> 2-fold) in CSE/PM10-exposed cells (Fig. 5B-E). Taken together, Nrf2
transcriptional activity and the expression of its key downstream genes
were enhanced in CSE/PM10-exposed BEAS-2B cells, and antioxidants
further promoted the Nrf2 activation and Nrf2-mediated gene expres-
sion.
3.6. SFN and SFNAC attenuate CSE- and PM10-induced MAPK signaling
activation
Mitogen-activated protein kinase (MAPK) is an essential regulator of
inflammatory mediators, including NO and pro-inflammatory cytokines
(Feng et al., 2002). Oxidative stress triggers the induction of tran-
scription factors, such as Nrf2 and NF-κB, via the MAPK signaling
pathway (Guo et al., 2015). To determine whether the relevance of the
MAPK pathway in the ability of SFN and SFNAC to suppress oxidative
stress and inflammation, we investigated the phosphorylation levels of
ERK, JNK, and p38 in CSE/PM10-exposed bronchial epithelial cells after
SFN/SFNAC treatment. While CSE/PM10 exposure activated ERK and
JNK, the phosphorylation levels of ERK and JNK decreased after
treatment with SFN or SFNAC (Fig. 6), suggesting a role of ERK and JNK
pathways in the protective effects of SFN and SFNAC in bronchial
epithelial cells exposed to CSE/PM10.
9
Toxicology in Vitro 67 (2020) 104883
4. Discussion
PM exposure causes various adverse health effects, including pul-
monary function insufficiency and exacerbation of chronic respiratory
and cardiovascular diseases, which often lead to emergency department
visits and hospitalization (Brunekreef and Holgate, 2002; Guaita et al.,
2011; Halonen et al., 2009). In addition, exposure to PM is strongly
associated with increased incidence of pulmonary diseases (Dockery
et al., 1993; Pope 3rd et al., 2002; Pope 3rd et al., 2004). Despite
epidemiologic evidence of the role of PM exposure in COPD exacerba-
tion and increased mortality (Gan et al., 2013; Ko et al., 2007; Thurston
et al., 2016), the mechanisms underlying PM-mediated COPD pro-
gression remain unclear.
PM is chemically diverse and induces inflammation and oxidative
stress in various cell types (Tao et al., 2003). Many recent studies have
assessed the effects of fine PM and cigarette smoke. One such study
reported that PM2.5 aggravated CSE-induced airway inflammation via
the ERK-Wnt5a pathway in a COPD model (Wang et al., 2019). Another
study showed that it enhanced cigarette-induced apoptosis in bronchial
epithelium (Zhou et al., 2019). Furthermore, ROS-mediated in-
flammation and upregulation of inflammatory cytokines, including IL-
6, IL-8, and TNF-α, have been implicated in PM10-induced cell toxicity
(Schins et al., 2004). In this study, we confirmed the adverse effects of
PM and CSE on inflammation and oxidative stress in bronchial epi-
thelial cells, as well as provided evidence on the alleviating effects of
the antioxidants SFN and SFNAC, which are mediated via Nrf2 and
MAPK pathways.
For our experiments, we selected the optimal PM type and con-
centration from preliminary experiments. In our preliminary experi-
ments, after treating BEAS-2B with 1% CSE for 7 days, we exposed them
to PM0.1, PM2.5, and PM10 at different concentrations (12.5–200 μg/mL)
and examined which PM and at which dose IL-6 and IL-8 expression
was most strongly induced. We found that PM10 (100 and 200 μg/mL)
was the most potent in enhancing CSE-mediated IL-6 and IL-8 pro-
duction in BEAS-2B cells. In this study, we used 100 μg/mL PM10,
which is equivalent to 13.8 μg/cm2 of the surface area of the cell culture
dishes used. An adult person inhales an average of 20 m3 of air per day.
Assuming a PM10 concentration of 150 μg/m3 by volume, which is the
24 h interim target-1 level according to the WHO air quality guidelines,
PM10 inhalation results in a daily deposition of 11.6 μg/cm2 in the
extrathoracic airways, as approximately 67–77% of inhaled PM10 is
deposited in the 200 cm2 area of the extrathoracic airways (Kutlar Joss
et al., 2017; Hussein et al., 2019). Therefore, the dose of PM10 used
herein resembles the deposition of PM10 in a human body according to
the WHO interim target-1 level for PM10.
Previous studies have demonstrated that oxidative stress can pro-
mote acute exacerbation of COPD (Kluchova et al., 2007). Alveolar
macrophages and blood neutrophils from patients with exacerbation of
COPD are activated and promote ROS secretion, particularly superoxide
radicals and hydrogen peroxide (Schaberg et al., 1995). Oxidative stress
and inflammation are important factors in the pathophysiology of
COPD. The most common method of establishing animal models to
study COPD is by CS inhalation. CS is the major risk factor for COPD,
leading to chronic airway inflammation. Therefore, CSE has been
widely used to identify the mechanisms by which CS induces lung in-
jury. We hypothesized that smoking aggravates PM-induced in-
flammation and oxidative stress in bronchial epithelial cells. To test this
hypothesis in a cell culture system, we chronically exposed BEAS-2B
cells to CSE, followed by acute exposure to PM10. In many studies that
have used CSE, variable concentrations and duration of exposure have
been applied to different cell types (Hoffmann et al., 2013; Krimmer
and Oliver, 2011; Lee et al., 2012; Li et al., 2007; van der Vaart et al.,
2004). In one study, oxidative stress-mediated inflammation and se-
nescence were observed in cells after moderate CSE exposure, whereas
programmed cell death was observed in cells after severe smoking stress
(Krimmer and Oliver, 2011). Although the inflammatory responses are
E.S. Son, et al.
induced in human bronchial epithelial cells both by short-term ex-
posure (up to 24 h) and long-term exposure (up to 6 months) to CSE
(Hoffmann et al., 2013; van der Vaart et al., 2004), in this study, we
chose to chronically expose cells to CSE, resembling the development of
COPD animal models by chronic CSE inhalation (more than a month).
In addition, we found that 1% CSE was the optimal concentration for
cell viability, considering a long-term CSE exposure (Fig. 1).
In our study, PM aggravated CSE-induced oxidative stress and in-
flammation in bronchial epithelial cells. Furthermore, the secretion of
pro-inflammatory cytokines induced by CSE exposure was significantly
increased when cells were treated with PM10. Exposure to CSE and
PM10 enhanced cytotoxicity and intracellular ROS generation in human
bronchial epithelial cells. Although PM10 impaired cell viability and
increased LDH release in CSE-exposed BEAS-2B cells, it did not cause
apoptotic cell death (Fig. S1).
Several studies have suggested that the cytotoxic and inflammatory
responses induced by CSE or PM10 are similar in that they increase
oxidative stress and are mediated by the MAP kinase pathway. NF-κB,
p38, JNK, and ERK 1/2, to some extent, are involved in CSE-induced
inflammation (Barnes, 2014; Hellermann et al., 2002; Lau et al., 2012;
Yoshida and Tuder, 2007). In addition, PM10-induced inflammation is
mediated by the AP-1, NF-κB, MAPK, and redox signaling pathways
(Donaldson, 2003; Montiel-Davalos et al., 2010; Wang et al., 2017). Mo
et al. (2012) reported that co-exposure to CSE and ultrafine particles
enhances inflammation in endothelial cells via p38 and ERK 1/2
phosphorylation (Mo et al., 2012). However, in the present study, we
found that the phosphorylation of ERK 1/2 and JNK increased in
bronchial epithelial cells after CSE and PM10 exposure.
Recently, exposure to CS and PM has been shown to cause oxidative
stress and inflammation. Antioxidant supplements have been proposed
as a method to alleviate the adverse effects of PM exposure on the
cardiorespiratory system. Tashakkor et al. (2011) demonstrated that
antioxidant supplements, such as Vitamin C and E, have protective ef-
fects against damage caused to human lungs by air pollution
(Tashakkor et al., 2011). They also found that omega-3 fatty acids
mitigated the adverse effects of PM on heart rate variability. Moreover,
probucol and antioxidant vitamins rescue CS-mediated defects in
ischemia-induced neovascularization by improving the function of en-
dothelial progenitor cells (Turgeon et al., 2010). Furthermore, SFN and
its metabolite SFN-NAC have antioxidant, anti-aging, and anti-in-
flammatory properties, and are able to block diesel exhaust particle-
induced oxidation stress and inflammation (Santin-Marquez et al.,
2019; Li et al., 2013). However, there is no documented evidence on the
beneficial effects of antioxidants in CSE and PM10-exposed bronchial
alveolar epithelial cells. In this study, we demonstrated that the anti-
oxidants SFN and SFNAC decreased ROS generation, cellular toxicity,
and inflammation in bronchial epithelial cells exposed to CSE and
PM10. However, SFN was more potent in regulating GSH, IL-6, and IL-8
levels in CSE and PM10 exposed BEAS-2B cells. Differences in their
conjugation status might have attributed to the differences in the bio-
logical responses. Although in physiological pH ranges, SFNAC dis-
sociates into SFN, this may not provide sufficient SFN amounts to affect
the GSH content (Conaway et al., 2001).
We also investigated the effects of SFN and SFNAC on Nrf2 signaling
and the expression of inflammation-related genes (Figs. S3 and S4). As
expected, cells treated with SFN or SFNAC expressed lower levels of
inflammatory genes compared to control CSE/PM-treated cells (Fig.
S3). The Nrf2 pathway, which is essential in immune responses initiated
by oxidative stress, was activated after treatment with antioxidants in
control cells (Fig. S4). Therefore, the protective effects of antioxidants
in CSE/PM10-treated cells are attributable to inflammation suppression
and Nrf2 signaling activation. Of course, it is not clear whether the
inhibitory effects of SFN and SFNAC on CSE/PM10 are due to in-
flammatory-related genes or side effects that are competing with PM10
and CSE. Therefore, future studies are required to uncover the me-
chanisms by which SFN or SFNAC represses CSE/PM10-induced
10
Toxicology in Vitro 67 (2020) 104883
inflammation and oxidative stress. Furthermore, the protective effects
of SFN and SFNAC against CSE and PM10 need to be confirmed in an-
imal models.
In conclusion, our findings suggest that the combination of CSE and
PM exacerbates oxidative stress and inflammation in bronchial epi-
thelial cells. Moreover, SFN and SFNAC attenuate CSE/PM10-induced
cytotoxicity and inhibit oxidative stress and inflammation via the ERK,
JNK, and Nrf2 signaling pathways. The better understanding of the
effects of PM10 and long-term CSE exposure in bronchial cells may lead
to the development of novel intervention strategies to prevent exacer-
bation of chronic pulmonary diseases.
Funding acknowledgments
This work was supported by the Gachon University Gil Medical
Center (FRD2015–13–02) and the Korea Research Foundation (NRF-
2017R1D1A1B03035535) (to SYK), (NRF-2018R1D1A1B07048747) (to
SHK), (NRF-2018R1A6A3A11047142) (to ESS).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.tiv.2020.104883.
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