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Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis

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The skin is the largest organ of the human body and the one mostly exposed to outdoor contaminants. To evaluate the biological mechanisms underlying skin damage caused by fine particulate matter (PM2.5), we analyzed the effects of PM2.5 on cultured human keratinocytes and the skin of experimental animals. PM2.5 was applied to human HaCaT keratinocytes at 50 µg/mL for 24 h and to mouse skin at 100 µg/mL for 7 days. The results indicate that PM2.5 induced oxidative stress by generating reactive oxygen species both in vitro and in vivo, which led to DNA damage, lipid peroxidation, and protein carbonylation. As a result, PM2.5 induced endoplasmic reticulum stress, mitochondrial swelling, and autophagy, and caused apoptosis in HaCaT cells and mouse skin tissue. The PM2.5-induced cell damage was attenuated by antioxidant N-acetyl cysteine, confirming that PM2.5 cellular toxicity was due to oxidative stress. These findings contribute to understanding of the pathophysiological mechanisms triggered in the skin by PM2.5, among which oxidative stress may play a major role.
PM2.5 induces ROS production leading to oxidative damage. Cells were treated with PM2.5 at the indicated concentrations for 24 h. a ROS generation was assessed by the H2DCFDA assay. b Apoptotic and necrotic cells were detected by Hoechst 33342 and PI nuclear staining, respectively. Arrows indicate apoptotic bodies and red color indicates necrotic cells. c Cells were treated with 50 µg/mL PM2.5 for the indicated times and ROS generation was assessed by the H2DCFDA assay. Cells were pre-treated with NAC (1 mM) for 1 h and then treated with PM2.5 (50 µg/mL) for 24 h. d ROS levels were assessed by confocal microscopy after H2DCFDA staining. e Cell viability was analyzed by trypan blue exclusion. Cells were treated with 50 µg/mL PM2.5f for the indicated times or g with the indicated concentrations of PM2.5 for 8 h and analyzed for the generation of 8-oxoG in DNA by avidin-TRITC binding using confocal microscopy. i DNA damage was evaluated by the Comet assay; representative images show comet tails and the graph shows quantification of cellular DNA damage. j Lipid peroxidation was analyzed by confocal microscopy after DPPP staining. k Protein oxidation was assayed by measuring carbonyl formation. l Zebrafish was pre-treated or not with NAC and then treated with PM2.5 (50 µg/mL) for 24 h, and analyzed for ROS production by H2DCFDA staining; zebrafish treated with H2O2 was used as a positive control. Mouse dorsal skin was treated with PM2.5 (100 µg/mL) for 7 days. m Tissue lysates were analyzed for H2A.X expression by immunoblotting; actin was used as loading control. n Immunohistochemistry of mouse tissue to analyze 4-HNE levels as a marker of lipid peroxidation; signals were detected in a peroxidase reaction (brown), and slides were counterstained with hematoxylin; magnification: ×400. o Protein carbonylation in mouse tissue was assessed using an immunohistochemical staining kit for protein carbonyls. *p < 0.05 compared to control groups and #p < 0.05 compared to PM2.5-treated groups. (Color figure online)
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Archives of Toxicology (2018) 92:2077–2091
https://doi.org/10.1007/s00204-018-2197-9
IN VITRO SYSTEMS
Particulate matter 2.5 damages skin cells byinducing oxidative stress,
subcellular organelle dysfunction, andapoptosis
MeiJingPiao1· MeeJungAhn2· KyoungAhKang1· YeaSeongRyu1· YuJaeHyun1· KristinaShilnikova1·
AoXuanZhen1· JinWooJeong3· YungHyunChoi3· HeeKyoungKang1· YoungSangKoh1· JinWonHyun1
Received: 9 November 2017 / Accepted: 21 March 2018 / Published online: 26 March 2018
© The Author(s) 2018
Abstract
The skin is the largest organ of the human body and the one mostly exposed to outdoor contaminants. To evaluate the
biological mechanisms underlying skin damage caused by fine particulate matter (PM2.5), we analyzed the effects of PM2.5
on cultured human keratinocytes and the skin of experimental animals. PM2.5 was applied to human HaCaT keratinocytes
at 50µg/mL for 24h and to mouse skin at 100µg/mL for 7days. The results indicate that PM2.5 induced oxidative stress
by generating reactive oxygen species both invitro and invivo, which led to DNA damage, lipid peroxidation, and protein
carbonylation. As a result, PM2.5 induced endoplasmic reticulum stress, mitochondrial swelling, and autophagy, and caused
apoptosis in HaCaT cells and mouse skin tissue. The PM2.5-induced cell damage was attenuated by antioxidant N-acetyl
cysteine, confirming that PM2.5 cellular toxicity was due to oxidative stress. These findings contribute to understanding of
the pathophysiological mechanisms triggered in the skin by PM2.5, among which oxidative stress may play a major role.
Keywords PM2.5· Oxidative stress· Apoptosis· Endoplasmic reticulum stress· Mitochondrial damage· Autophagy
Introduction
Global air pollution has become a major threat to human
health. This worldwide problem is especially relevant to
the release of fine particulate matter (PM2.5), which has the
aerodynamic diameter less than 2.5µm and originates from
incomplete coal combustion and diesel vehicle exhaust in
Korea (Lee etal. 2005; Jung etal. 2017). In recent years,
the relationship between PM2.5 production and public health
hazards has attracted an increasing attention. Several toxi-
cological and epidemiological studies have suggested that
PM2.5 exerts negative biological effects on several major
organs, including the lung (Liu etal. 2017), immune system
(Zhao etal. 2013), cardiovascular system (Du etal. 2016),
and nervous system (Wang etal. 2017). Among the affected
organs, the skin is the primary tissue exposed to ambient
pollutants and, similar to the respiratory tract, presents an
interface between the body and surrounding atmosphere.
PM2.5-carrying organic chemicals such as polycyclic aro-
matic hydrocarbons (PAHs) are highly lipophilic and easily
penetrate the skin (Krutmann etal. 2014). PAHs are potent
activators of the aryl hydrocarbon receptor (AhR), which is a
ligand-dependent transcription factor expressed in keratino-
cytes and melanocytes (Fritsche etal. 2007; Jux etal. 2011).
AhR activation by PAHs upregulates the expression of
cytochrome P450 (CYP1A1) involved in the metabolism
of xenobiotics (Vogel etal. 2016) and promotes generation
of intracellular reactive oxygen species (ROS) (Costa etal.
2010). Accumulated evidence indicates that oxidative stress
is a common mechanism of PM2.5-induced damage (Gual-
tieri etal. 2012; Kouassi etal. 2010). Recently, the effect
of PM2.5 on the skin has attracted attention of both clinical
dermatologists and basic scientists (Han etal. 2016; Li etal.
2017), who recognized ambient PM2.5 as a crucial risk fac-
tor in skin diseases. Thus, PM2.5 was shown to aggravate
symptoms in children with allergic dermatitis and eczema
(Song etal. 2011), and to promote inflammatory disorders,
* Jin Won Hyun
jinwonh@jejunu.ac.kr
1 Jeju National University School ofMedicine andJeju
Research Center forNatural Medicine, Jeju63243,
RepublicofKorea
2 Laboratory ofVeterinary Anatomy, College ofVeterinary
Medicine, Jeju National University, Jeju63243,
RepublicofKorea
3 Department ofBiochemistry, College ofOriental Medicine,
Dongeui University, Busan47340, RepublicofKorea
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2078 Archives of Toxicology (2018) 92:2077–2091
1 3
aging, androgenetic alopecia, and cancers of the skin (Kim
etal. 2016).
Mitochondria are unique double-membrane subcellular
organelles that provide energy through oxidative phospho-
rylation and participate in metabolic and genetic processes in
the body. Once mitochondria are disrupted, their dysfunction
leads to reduced generation of ATP and higher production of
ROS. Mitochondria are targeted by environmental pollutants
such as PM2.5 (Guo etal. 2017) and mitochondrial dam-
age may be a critical part of the pathophysiological mecha-
nisms induced by PM2.5 exposure. Oxidative stress has been
shown to be an initiator and major contributor to both endo-
plasmic reticulum (ER) stress (Hotamisligil 2010; Kaneto
etal. 2005) and lysosome-mediated autophagy (Lee etal.
2012); however, the research on molecular pathways linking
atmospheric PM2.5 and skin damage is limited. Although
skin is the organ mostly exposed to PM2.5, the association
of skin-damaging effects of PM2.5 with oxidative stress and
dysfunction of subcellular organelles such as mitochondria,
ER, and lysosomes is still not fully elucidated. In this study,
we investigated the effects of PM2.5 on the induction of oxi-
dative stress and structure of subcellular organelles using
invitro and invivo models and explored the mechanisms
underlying PM2.5 toxicity for the skin.
Materials andmethods
Preparation of PM2.5
Diesel particulate matter NIST 1650b (PM2.5) was purchased
from Sigma-Aldrich, Inc. (St. Louis, MO, USA). PM2.5 stock
solution (25mg/mL) was prepared in dimethyl sulfoxide
(DMSO) and sonicated for 30min to avoid agglomeration of
the suspended PM2.5 particles. Experiments were performed
within 1h of stock preparation to avoid variability in PM2.5
composition in solution.
Cell culture
Human HaCaT keratinocytes (Amore Pacific Company,
Gyeonggi-do, Republic of Korea) were maintained at 37°C
in an incubator with a humidified atmosphere of 5% CO2.
Cells were cultured in Dulbecco-modified Eagle medium
(DMEM) containing 10% heat-inactivated fetal bovine
serum and antibiotic–antimycotic (100units/mL penicillin,
100µg/mL streptomycin, and 0.25µg/mL amphotericin B)
(Gibco, Life Technologies Co., Grand Island, NY, USA).
Animal experiment
In vivo experiments were conducted using HR-1 hairless
male mice (OrientBio, Kyungki-do, Republic of Korea)
in accordance with the guidelines for the care and use of
laboratory animals at Jeju National University (Jeju, Repub-
lic of Korea) (permit number: 2017-0026). Mice were
randomly divided into three groups (n = 4 each): control,
and treated with PM2.5 or N-acetyl cysteine (NAC; Sigma-
Aldrich) + PM2.5. PM2.5 was dispersed in propylene glycol to
the concentration of 100µg/mL, spread on a nonwoven poly-
ethylene pad over a 1cm2 area, and applied to the dorsal skin
of mice for 7 consecutive days. At the end of the treatment,
the exposed skin tissue was immediately dissected for his-
tological and biochemical analysis as previously described
(Lee etal. 2016).
ROS measurement
Cells were incubated with different concentrations of PM2.5
(25, 50, 75, and 100µg/mL) for 24h or treated with PM2.5at
a concentration of 50µg/mL for different times (1, 2, 4, 8,
12, and 24h). After staining with 25µM 2,7-dichlorodi-
hydrofluorescein diacetate (H2DCFDA; Molecular Probes,
Eugene, OR, USA) dye for 10min, H2DCF fluorescence
was detected by flow cytometry (Becton Dickinson, Moun-
tain View, CA, USA) and analyzed using the Cell Quest
software. For imaging, cells were plated in a 4-well glass
chamber slide, treated with 1mM NAC and/or 50µg/mL
PM2.5, and analyzed for intracellular and mitochondrial
ROS production after staining with H2DCFDA and dihy-
drorhodamin 123 (DHR 123; Molecular Probes), respec-
tively, for 30min. Images of stained cells were generated by
confocal microscopy as previously described (Kim and Yoo
2016; Soeur etal. 2017). To detect ROS in zebrafish treated
with NAC and/or PM2.5, they were incubated with 10µM
H2DCFDA for 30min in the dark at 28.5°C. After anes-
thesia with 1-phenoxy-2-propanol (1/500 dilution; Acros
Organics, Morris Plains, NJ, USA), the stained zebrafish
were observed under a fluorescence microscope (Zeiss
AX10, Carl Zeiss, Göttingen, Germany) (Jeong etal. 2017).
Hoechst 33342/propidium iodide (PI) staining
Cells were treated with different concentrations of PM2.5 (25,
50, 75, and 100µg/mL) for 24h or pre-treated with 1mM
NAC for 1h and then treated with 50µg/mL of PM2.5 for
24h and then stained with DNA-specific fluorescent dyes
Hoechst 33342 (10µM) and propidium iodide (PI; 5µg/mL)
(both from Sigma-Aldrich). Cells with fragmented nuclei
stained with Hoechst 33342 were considered apoptotic and
those stained with PI were considered necrotic. Cell staining
was visualized under a fluorescence microscope equipped
with a CoolSNAP-Pro color digital camera (Media Cyber-
netics, Rockville, MD, USA) and the proportions of apop-
totic and neurotic cell were quantified.
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2079Archives of Toxicology (2018) 92:2077–2091
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Trypan blue assay
Cells were seeded in 35-mm culture dishes at a concentra-
tion of 1.0 × 105 per mL, cultured for 16h, and treated with
different concentrations of PM2.5 (25, 50, 75, and 100µg/
mL) for 24h. Then, 5µL of 0.1% trypan blue solution was
added to 0.1mL cell suspension for 5min at room tempera-
ture, and the numbers of viable and dead cells were deter-
mined under a microscope using 10× magnification. Cell
viability (%) was calculated as: unstained cells/(unstained
cells + stained cells) × 100%.
Detection of8‑oxoguanine
Cellular DNA was isolated using the G-DEX™ IIc Genomic
DNA Extraction Kit (iNtRON Biotechnology, Inc., Sung-
nam, Kyungki-Do, Republic of Korea) and quantified by
spectrophotometry. The amount of 8-hydroxy-2-deoxy-
guanosine (8-OHdG, the deoxyriboside form of 8-oxoG) in
DNA was determined using the BIOXYTECH® 8-OHdG-
EIA™ kit (OXIS Health Products, Inc., Portland, OR, USA)
according to the manufacturer’s instructions. The amount of
8-oxoG was also estimated by a fluorescence-binding assay:
cells were fixed and permeabilized with ice-cold methanol
for 15min and 8-oxoG was visualized with avidin-TRITC
conjugate (Sigma-Aldrich) under a confocal microscope
(Piao etal. 2011).
Single cell gel electrophoresis (Comet assay)
Cell-coated slides were immersed in lysis buffer (2.5M
NaCl, 100mM Na-EDTA, 10mM Tris, 1% Trion X-100,
and 10% DMSO, pH 10) for 1h at 4°C, subjected to elec-
trophoresis, stained with ethidium bromide, and observed
under a fluorescence microscope equipped with an image
analysis software (Kinetic Imaging, Komet 5.5, UK) as pre-
viously described (Park etal. 2017). The percentage of the
total fluorescence in the comet tail and the length of the tail
was recorded in 50 cells per slide.
Lipid peroxidation assay
Cells were stained with 5µM of a fluorescent probe diphe-
nyl-1-pyrenylphosphine (DPPP; Molecular Probes) as
described (Morita etal. 2016) and analyzed using an Olym-
pus FV1200 laser scanning microscope equipped with the
FV10-ASW viewer 4.2 software. Mouse skin tissue was
analyzed by immunohistochemistry using an antibody to
4-hydroxy-2-nonenal (4-HNE) (Cosmo Bio Co., Tokyo,
Japan).
Protein carbonylation
Total cellular proteins were extracted with lysis buffer and
quantified by spectrophotometry, and protein carbonyla-
tion was determined using an OxiSelect™ protein carbonyl
ELISA kit (Cell Biolabs, San Diego, CA, USA) according
to the manufacturer’s instructions. In tissues, protein carbon-
ylation was assessed using an immunohistochemical staining
kit (Cosmo Bio Co.)
Western blotting
Cell and mouse skin lysates were subjected to SDS-PAGE,
and the separated proteins were transferred to membranes
and incubated with primary antibodies against phospho-
H2A.X (Ser139), CHOP, phospho-PERK, beclin-1, LC3B,
caspase-3, and caspase-9 (Cell Signaling Technology, Bev-
erly, MA, USA), GRP78, Bax (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), phospho-IRE1 (Abcam, Cambridge,
MA, USA), and actin (Sigma-Aldrich) followed by the
incubation with a secondary antibody (Pierce, Rockford,
IL, USA). Protein bands were detected using an Amersham
Enhanced Chemiluminescence Plus Western Blotting Detec-
tion system (GE Healthcare Life Sciences, Buckingham-
shire, UK).
ER staining
Cells were reacted with an ER-tracker blue-white DPX dye
(Molecular Probes) for 30min, and images were taken under
a confocal microscope (Li etal. 2015).
Quantification of Ca2+ level
Cells were loaded for 30min with 10µM fluo-4-acetoxy-
methyl ester (Fluo-4-AM) or with Rhod-2 acetoxymethyl
ester (Rhod-2 AM) (Molecular Probes) to detect intracellular
and mitochondrial Ca2+, respectively, and fluorescence was
measured by confocal microscopy (Wang etal. 2016).
Mitochondrial membrane potential (Δψm)
measurement
Mitochondrial Δψ was analyzed by confocal micros-
copy after staining with 5,5,6,6-tetrachloro-1,1,3,3-
tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Invitro-
gen, Carlsbad, CA, USA), a lipophilic cationic fluorescence
dye (Yao etal. 2016).
Acridine orange staining
To analyze autophagy, cells were reacted with acrid-
ine orange (Invitrogen) for 15min and fluorescence was
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2080 Archives of Toxicology (2018) 92:2077–2091
1 3
measured using a fluorescence microscope (BH2-RFL-T3;
Olympus, Tokyo, Japan) (Farah etal. 2016). Depending
on the acidity, autophagic lysosomes appeared as orange/
red fluorescent cytoplasmic vesicles, while the nuclei were
stained green.
LC3 transfection anddetection ofpunctate
LC3‑positive structures
Membrane-bound microtubule-associated protein 1 light
chain 3 (LC3) is present in the autophagic double-membrane
structure, which is an important marker of autophagy (Fazeli
and Wehman 2017). Cells were transfected with GFP-tagged
LC3 using Lipofectamine reagent (Invitrogen) according to
the manufacturer’s instructions and GFP-LC3 fluorescence
was observed under a confocal microscope.
Histological analysis
Skin pieces were fixed in 4% paraformaldehyde, embed-
ded in paraffin, and cut into 5µm sections, which were then
deparaffinized and stained with hematoxylin and eosin. The
height of epidermis (from the stratum basale to the stratum
corneum) was measured in ten randomly chosen fields from
three representative sections per group by microscopy at
100× magnification using a digital camera. Immunohisto-
chemistry was performed by incubating skin sections with
primary anti-Bax antibody (1:400; Abcam, Cambridge, MA,
USA) for 1h and the reaction was visualized using an ABC
Elite kit (Vector Labs, Burlingame, CA, USA). The sections
were counterstained with hematoxylin before mounting. For
the insitu detection of apoptotic cells in skin sections, the
DeadEnd colorimetric TUNEL system (Promega, Wiscon-
sin, WI, USA) was used according to the manufacturer’s
recommendation.
Transmission electron microscopy (TEM)
Cells and tissues were fixed, dehydrated, incubated with
increasing concentrations of propylene oxide dissolved in
ethanol, and infiltrated with increasing concentrations of
Eponate 812 resin. Samples were baked in a 65°C oven
overnight, sectioned in an ultramicrotome, and examined
by TEM using a field electron emission unit (JEM-2100F,
JEOL) at the Korean Basic Science Institute (Chuncheon,
Republic of Korea).
Statistical analysis
Statistical significance of the difference between groups
was determined by analysis of variance and Tukey’s tests
using the SigmaStat version 3.5 software (Systat Software
Inc., San Jose, CA, USA). All data are presented as the
mean ± standard error. p < 0.05 was considered to indicate
statistically significant difference.
Results
PM2.5 induced oxidative stress bothinvitro
andinvivo
To investigate the potential role of oxidative stress
induced by PM2.5, we measured ROS generation and cel-
lular damage in PM2.5-treated human keratinocytes and
mouse skin. Figure1a shows that PM2.5 treatment pro-
moted the production of ROS in a dose-dependent manner
as evidenced by H2DCFDA staining. Analysis of Hoechst
33342/PI staining indicated that PM2.5at a concentration
of 50µg/mL induced apoptosis (Hoechst 33342-stained
cells); however, at concentrations above 75µg/mL, PM2.5
induced necrosis (PI-stained cells) (Fig.1b). We used
50µg/mL PM2.5 as the optimal concentration in further
experiments. ROS generation was increased starting from
1h up to 24h of treatment with 50µg/mL PM2.5 (Fig.1c).
Next, we determined whether PM2.5 induced cytotoxicity
via ROS generation. An antioxidant NAC was not toxic
for HaCaT cells at concentrations up to 1mM (MTT test;
data not shown); therefore, 1mM NAC was used in this
study. Confocal microscopy indicated that green fluores-
cence was increased in PM2.5-treated cells compared to
control, but the effect was suppressed by 1mM NAC
(Fig.1d), indicating that PM2.5 stimulated ROS produc-
tion in keratinocytes. Furthermore, PM2.5 induced cyto-
toxicity as evidenced by trypan blue exclusion; however,
1mM NAC reduced it (Fig.1e), suggesting that PM2.5
caused cytotoxicity via ROS. We next evaluated the dam-
age of intracellular macromolecules by PM2.5. The level of
8-oxoG, a hallmark of oxidative DNA damage, was meas-
ured based on 8-OHdG detection. The results indicated
that PM2.5 promoted the generation of 8-oxoG in DNA in
a time-dependent manner when used at 50µg/mL (Fig.1f)
and in a dose-dependent manner when administered for
8h (Fig. 1g). In addition, condensed 8-oxoG staining
was observed in PM2.5-treated cells, whereas 1mM NAC
reduced it (Fig.1h). The Comet assay assessing DNA
breakage indicated that PM2.5 increased the presence of
cellular DNA tails by 30% compared to control; however,
NAC pre-treatment reduced it to 12% (Fig.1i). Further-
more, fluorescence intensity of DPPP oxide, an indicator
of lipid peroxidation, was enhanced in PM2.5-incubated
cells but significantly reduced by NAC pre-treatment
(Fig.1j). Similarly, the level of protein carbonylation,
a biomarker of oxidative stress-induced protein damage,
was increased in PM2.5-treated cells, whereas NAC could
prevent PM2.5-induced carbonyl formation (Fig.1k). To
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2081Archives of Toxicology (2018) 92:2077–2091
1 3
a b
c
0
100
200
300
Intracellular ROS [%]
PM2.5
0 1 2 4 8 12 24 h
*
*
* * *
*
0
200
400
600
Intracellular ROS [%]
PM2.5 [μg/mL]
*
*
* *
0 25 50 75 100
ControlPMNAC+PM
e
d
0
30
60
90
120
*
#
Control PM2.5 NAC+PM2.5
Cell viability
0
10
20
30
Index of ROS generation
Control PM2.5 NAC+PM2.5
*
#
Control PM2.5NAC+PM2.5
DCF-DA
0
20
40
60
80
100
8-OHdG level (ng/ml
* * * *
*
PM
2.5
0 1 4 8 12 24 h
f
0 25 50 75 100
PM2.5 [μg/mL]
Index of apoptotic
& necrotic cells
0
10
20
30
40
50
Apoptotic cells
Necrotic cells
PM2.5 [μg/mL]
0 25 50 75 100
*
**
*
*
*
**
Fig. 1 PM2.5 induces ROS production leading to oxidative damage. Cells were
treated with PM2.5 at the indicated concentrations for 24h. a ROS generation
was assessed by the H2DCFDA assay. b Apoptotic and necrotic cells were
detected by Hoechst 33342 and PI nuclear staining, respectively. Arrows indi-
cate apoptotic bodies and red color indicates necrotic cells. c Cells were treated
with 50µg/mL PM2.5 for the indicated times and ROS generation was assessed
by the H2DCFDA assay. Cells were pre-treated with NAC (1mM) for 1h and
then treated with PM2.5 (50 µg/mL) for 24h. d ROS levels were assessed by
confocal microscopy after H2DCFDA staining. e Cell viability was analyzed by
trypan blue exclusion. Cells were treated with 50µg/mL PM2.5 f for the indi-
cated times or g with the indicated concentrations of PM2.5 for 8h and ana-
lyzed for the generation of 8-oxoG in DNA by avidin-TRITC binding using
confocal microscopy. i DNA damage was evaluated by the Comet assay; rep-
resentative images show comet tails and the graph shows quantification of cel-
lular DNA damage. j Lipid peroxidation was analyzed by confocal microscopy
after DPPP staining. k Protein oxidation was assayed by measuring carbonyl
formation. l Zebrafish was pre-treated or not with NAC and then treated with
PM2.5 (50 µg/mL) for 24h, and analyzed for ROS production by H2DCFDA
staining; zebrafish treated with H2O2 was used as a positive control. Mouse
dorsal skin was treated with PM2.5 (100µg/mL) for 7 days. m Tissue lysates
were analyzed for H2A.X expression by immunoblotting; actin was used as
loading control. n Immunohistochemistry of mouse tissue to analyze 4-HNE
levels as a marker of lipid peroxidation; signals were detected in a peroxidase
reaction (brown), and slides were counterstained with hematoxylin; magnifi-
cation: ×400. o Protein carbonylation in mouse tissue was assessed using an
immunohistochemical staining kit for protein carbonyls. *p < 0.05 compared to
control groups and #p < 0.05 compared to PM2.5-treated groups. (Color figure
online)
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2082 Archives of Toxicology (2018) 92:2077–2091
1 3
validate these results invivo, we used a zebrafish model.
As shown in Fig.1l, PM2.5 treatment also upregulated
ROS production in zebrafish, which was attenuated by
NAC. Finally, the results were confirmed in a mouse
model, which showed that PM2.5 treatment induced DNA
damage as indicated by the expression of phosphoryl-
ated histone H2A.X (Fig.1m), and stimulated lipid per-
oxidation (Fig.1n) and protein carbonylation (Fig.1o)
in mouse skin. Overall, these findings show that PM2.5
induced oxidative stress by enhancing ROS production,
which resulted in the damage of cellular components.
PM2.5‑induced oxidative damage caused ER stress
We next investigated whether PM2.5 oxidative effects resulted
in ER stress. PM2.5-treated cells were stained bright blue by
the ER-Tracker Blue-White DPX, indicating the induction of
ER stress which was attenuated by NAC (Fig.2a), suggest-
ing that PM2.5 promoted ER stress through ROS generation.
ER is a major intracellular Ca2+ reservoir, and disruption of
Ca2+ homeostasis activates ER stress (Jakobsen etal. 2008;
Xu etal. 2005). Confocal microscopy analysis revealed higher
intensity of Ca2+ fluorescence in PM2.5-treated cells compared
g h
0
10
20
30
40
50
% Fluorescence in tail
Control PM2.5 NAC+PM2.5
*
#
ControlPM2.5NAC+PM2.5
i
Avidin-TRITC
Control PM2.5NAC+PM2.5
0
30
60
90
120
8-OHdG level (ng/ml
* * *
PM2.5 [μg/mL]
0 25 50 75 100
0
1
2
3
4
5
Index of fluorescence intensity
Control PM2.5 NAC+PM2.5
*
#
j
DPPP
ControlPM2.5NAC+PM2.5
0
2
4
6
Index of fluorescence
intensity
Control PM2.5 NAC+PM2.5
*
#
k
0
2
4
6
8
Control PM
2.5
NAC+PM
2.5
*
#
Protein
carbonyl [nmol]
Fig. 1 (continued)
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2083Archives of Toxicology (2018) 92:2077–2091
1 3
with control, but the effect was reduced by NAC (Fig.2b). ER
stress promotes the expression of C/EBP homologous protein
(CHOP), which mediates apoptosis (Nishitoh 2012), and of the
ER chaperone and signaling regulator GRP78, which activates
protein kinase R-like ER kinase (PERK) through phospho-
rylation. In turn, phospho-PERK causes inhibition of transla-
tion and protein synthesis observed after ER stress (Bertolotti
etal. 2000). As shown in Fig.2c, PM2.5 induced the expres-
sion of ER stress-related proteins, including CHOP, GRP78,
phospho-PERK, and phospho-serine/threonine-protein kinase/
endoribonuclease inositol-requiring enzyme 1 (IRE1) in a
time-dependent manner. To confirm these results, we exam-
ined the induction of ER stress markers in mice treated with
PM2.5 and found that the expression of GRP78 and CHOP was
significantly increased in PM2.5-treated skin compared with
control; however, NAC reversed the effect (Fig.2d). These
observations were consistent with those invitro, suggesting
that PM2.5-induced ER stress may be associated with oxida-
tive stress.
PM2.5‑induced oxidative stress promoted
mitochondrial damage
Confocal microscopy analysis showed that, in cultured
human keratinocytes, ROS generation and Ca2+ overload
in mitochondria were enhanced by PM2.5 treatment but
reduced by NAC (Fig.3a, b). Mitochondrial membrane per-
meability is associated with apoptosis through the release
of cytochrome c and caspase activation (Chaudhary etal.
2016). A membrane-permeant dye JC-1 is widely used in
apoptosis studies to monitor the status of mitochondria
where JC-1 accumulates in a potential-dependent manner
as indicated by a fluorescence emission shift from green
(~ 529nm) to red (~ 590nm). Accordingly, mitochondrial
polarization (healthy state) or depolarization (damaged state)
can be revealed by an increase or decrease, respectively, in
the red/green fluorescence intensity ratio (Lee etal. 2017).
Confocal microscopy images showed that in control cells,
mitochondria exhibited strong red JC-1 fluorescence indica-
tive of Δψm polarization, which was reduced in PM2.5-treated
cells where green fluorescence indicative of Δψm depolariza-
tion was increased; however, the effect was suppressed by
NAC (Fig.3c).
It is known that proteins of the Bcl-2 family regulate
apoptosis by controlling mitochondrial permeability. There-
fore, we next examined whether PM2.5 affected the expres-
sion of Bax, a pro-apoptotic member of the Bcl-2 family.
The results indicated that PM2.5 increased Bax levels in
cultured human keratinocytes (Fig.3d) as well as in mouse
skin (Fig.3e, f); however, NAC pre-treatment prevented
l
MergePhaseDCF-DA
Normal
PM2.5
NAC+PM2.5
H2O2
m
Phospho-H2A.X
Actin
Normal PM2.5NAC+PM2.5
WB
n o
0
50
100
150
200
250
Fluorescence intensity
Normal PM2.5NAC+PM2.5 H2O2
*
#
*
Normal PM2.5NAC+PM2.5
Scale bar=20 μm Scale bar=20 μm
Normal PM2.5NAC+PM2.5
0
2
4
6
8
Normal PM2.5NAC+PM2.5
*
#
Relative expression level
(phospho-H2A.X/Actin)
Fig. 1 (continued)
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2084 Archives of Toxicology (2018) 92:2077–2091
1 3
PM2.5-induced upregulation of Bax expression (Fig.3e, f).
Cumulatively, these data indicate that PM2.5 increased oxida-
tive stress in mitochondria by stimulating ROS production,
which resulted in mitochondrial damage.
PM2.5‑induced oxidative stress caused autophagy
We next determined whether PM2.5-induced oxidative
stress could promote autophagy. In cultured keratinocytes,
Fig. 2 PM2.5 induces ER stress
via ROS generation. a, b Cells
were pre-treated or not with
NAC (1mM) for 1h and then
with PM2.5 (50µg/mL) for
24h and analyzed by confocal
microscopy for ER stress using
a ER-Tracker Blue-White DPX
staining and for intracellular
Ca2+ levels using b Fluo-4-AM
staining. Representative confo-
cal images are shown. Lysates
of c cells and d mouse skin
tissue were analyzed for the
expression of CHOP, GRP78,
phospho-PERK, and phospho-
IRE1 by western blotting. Actin
was used as loading control.
*p < 0.05 compared to control
groups and #p < 0.05 compared
to PM2.5-treated groups
Control PM2.5NAC+PM2.5
ER tracker
Control PM2.5NAC+PM2.5
Fluo-4
a
b
c
CHOP
PM
2.5
0 3 6 12 24 h
GRP78
Phospho-IRE1
Actin
Phospho-PERK
WB
Relative expression levels
0
1
2
3
4
*
CHOP/Actin
0
2
4
6
*
GRP78/Actin
0
1
2
3
4
*
*
Phospho-PERK/Actin
0
1
2
3
*
PM2.5
0 3 6 12 24 h
Phospho-IRE1α/Actin
0
1
2
3
4
Index of fluorescence intensity
Control PM2.5 NAC+PM2.5
*
#
0
4
8
12
16
Index of fluorescence intensity
Control PM2.5 NAC+PM2.5
*
#
d
CHOP
GRP78
Actin
Normal PM2.5NAC+PM2.5
WB
0
1
2
3
4
*
CHOP/Actin
#
0
2
4
6
8
10
Normal PM
2.5
NAC+PM
2.5
*
#
GRP78/Acti
n
Relative expression levels
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2085Archives of Toxicology (2018) 92:2077–2091
1 3
Fig. 3 PM2.5 induces mitochon-
drial damage via ROS genera-
tion. Cells were pre-treated or
not with NAC (1mM) for 1h
and then with PM2.5 (50µg/
mL) for 24h and analyzed by
confocal microscopy to assess
a mitochondrial ROS (DHR123
staining), b mitochondrial Ca2+
levels (Rhod-2 AM staining),
and c Δψm (JC-1 staining).
Lysates of d cells and e mouse
skin tissue were analyzed for
the expression of Bax protein
by western blotting. f Mouse
skin tissue was analyzed for Bax
expression by immunohisto-
chemistry. Nuclei were stained
with hematoxylin; arrows indi-
cate Bax. *p < 0.05 compared
to control groups and #p < 0.05
compared to PM2.5-treated
groups
a b
DHR 123
ControlPM2.5NAC+PM2.5
c
d PM2.5
0 3 6 12 24 h
Bax
Acti
n
WB
0
2
4
6
8
Relative expression
level (Bax/Actin)
*
PM2.5
0 3 6 12 24 h
0
10
20
30
Index of fluorescence intensity
Control PM2.5 NAC+PM2.5
*
#
0.0
0.5
1.0
1.5
2.0
2.5
Red/green fluorescence ratio
Control PM2.5 NAC+PM2.5
*
#
ControlPM2.5 NAC+PM2.5
Merge Depolarized Polarized
Δψm
0
2
4
6
8
Index of fluorescence intensity
Control PM2.5 NAC+PM2.5
*
#
Rhod-2 AM
Control PM2.5NAC+PM2.5
e
0
1
2
3
Normal PM
2.5
NAC+PM
2.5
*
#
Relative
expression level
(Bax/Actin)
Bax
Actin
WB
Normal PM2.5NAC+PM2.5f Normal PM2.5
NAC+PM2.5Negative control
Scale bar=20 μm
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2086 Archives of Toxicology (2018) 92:2077–2091
1 3
PM2.5 triggered accumulation of intracellular vacuoles
indicative of autophagy, as evidenced by staining with a
lysosome marker dye acridine orange (Fig.4a). The two
distinct steps of autophagy, autophagosome formation
and autolysosome formation, can be discerned by the pres-
ence of LC3-phospholipid conjugates (Tanida etal. 2005).
PM2.5-treated GFP-LC3-transfected cells had increased
levels of GFP-LC3-positive puncta (Fig.4b). In addition,
PM2.5 upregulated the expression of beclin-1, the protein
initiating autophagosome formation during autophagy, and
LC3B-II, the processed form of LC3, in a time-dependent
manner (Fig.4c). These invitro results were confirmed in
PM2.5-treated mouse skin (Fig.4d). However, the effects
of PM2.5 both in cultured cell and animals were reversed
by NAC (Fig.4a, b, d), suggesting that PM2.5 increased
autophagy through oxidative stress.
PM2.5‑induced oxidative stress promoted apoptotic
cell death
PM2.5 induced apoptosis both in cultured cells and mouse
skin tissues, as shown by the formation of apoptotic bodies
and DNA fragmentation revealed by Hoechst 33342 staining
and TUNEL assay, respectively; however, NAC pre-treat-
ment diminished the effects (Fig.5a, b). Another evidence
that PM2.5 promoted apoptosis was time-dependent increase
in the expression of cleaved caspase-9 and caspase-3
(Fig.5c), which indicated caspase activation in response
to mitochondrial membrane disruption. Similar results
were obtained for the mouse skin, where cleaved forms of
caspase-3 and caspase-9 were upregulated in response to
PM2.5 treatment; however, the effect was attenuated by NAC
(Fig.5d), suggesting that PM2.5 induced apoptosis via oxida-
tive stress.
PM2.5 internalization damaged theultrastructure
ofmouse skin tissue
TEM analysis revealed PM2.5 internalization in HaCaT
cells after 24h of exposure to 50µg/mL PM2.5 (Fig.6a-
2). In addition, to evaluate organelle ultrastructure in skin
cells following PM2.5 exposure, we performed TEM analy-
sis of mouse skin tissue after treatment with 100µg/mL
PM2.5. Compared to normal mice (Fig.6b-1), skin tissue
Fig. 4 PM2.5 induces autophagy
via ROS generation. Cells
were pre-treated or not with
NAC (1mM) for 1h, treated
with PM2.5 (50µg/mL) for
24h. a Cells were stained with
acridine orange and analyzed
for autophagy by fluorescence
microscopy. b Cells were
transfected with the GFP-
LC3 expression construct and
observed under a fluorescence
microscope. Lysates extracted
from c cells and d mouse skin
tissue were analyzed for the
expression of beclin-1 and
LC3B-II proteins by western
blotting; actin was used as load-
ing control. *p < 0.05 compared
to control groups and #p < 0.05
compared to PM2.5-treated
groups. (Color figure online)
a
c
b
AO
ControlPM2.5NAC+PM2.5
d
GFP-LC3
ControlPM2.5NAC+PM2.5
0.0
0.5
1.0
1.5
2.0
2.5
Beclin-1/Actin
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
LC3B-II/Actin
*
*
PM
2.5
0 3 6 12 24 h
Relative expression levels
*
*
0
1
2
3
*
#
Beclin-1/Actin
Relative expression levels
0
1
2
3
4
*
#
LC3B-II/Actin
Normal PM2.5NAC+PM2.5
Beclin-1
Actin
Normal PM2.5NAC+PM2.5
WB
LC3B-ϩ
PM2.5
0 3 6 12 24 h
Beclin-1
Actin
WB
LC3B-ϩ
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2087Archives of Toxicology (2018) 92:2077–2091
1 3
of PM2.5-treated mice showed increased swelling of mito-
chondria (Fig.6b-2) and ER (Fig.6b-3), and autophago-
some formation (Fig.6b-4), indicating that PM2.5 dis-
rupted intracellular network in the skin.
Discussion
According to the Air Korea site of Korea Environment Cor-
poration (2015, 2016, 2017) of the National Environmental
Research Institute of the Republic of Korea, the average
a
0
4
8
12
16
20
Index of apoptotic body
Control PM
2.5
NAC+PM
2.5
*
#
Control PM
2.5
NAC+PM
2.5
Hoechst 33342
c
PM
2.5
0 3 6 12 24 h
Actin
Cleaved caspase-3
Cleaved caspase-9
WB
Cleaved caspase-3
Actin
Cleaved caspase-9
Normal PM
2.5
NAC+PM
2.5
WB
d
b
Normal PM
2.5
NAC+PM
2.5
Scale bar=20 μm
0
1
2
3
*
#
Cleaved
caspase-9/Actin
0
1
2
3
4
*
#
Cleaved
caspase-3/Acti
n
Normal PM
2.5
NAC+PM
2.5
Relative expression levels
0
1
2
3
4
5
Cleaved
caspase-3/Actin
*
*
*
PM
2.5
0 3 6 12 24 h
0
1
2
3
4
Cleaved
caspase-9/Actin
*
Relative expression levels
Fig. 5 PM2.5 induces apoptosis via ROS generation. a Cells were pre-
treated or not with NAC (1mM) for 1h, treated with PM2.5 (50µg/
mL) for 24h, and analyzed for apoptotic body formation after Hoe-
chst 33342 staining; apoptotic bodies are indicated by arrows. b
Mouse skin treated with NAC and PM2.5 (100µg/mL) for 7 days was
analyzed for apoptosis by TUNEL staining; TUNEL-positive cells are
indicated by arrows. Lysates extracted from c cells and d mouse skin
tissue were analyzed for the expression of caspase-3 and caspase-9 by
western blotting. *p < 0.05 compared to control groups and #p < 0.05
compared to PM2.5-treated groups
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2088 Archives of Toxicology (2018) 92:2077–2091
1 3
concentrations of PM2.5 in the air of seven major Korean
cities from January to March were 31, 28, and 29µg/m3 in
2015, 2016, and 2017, respectively, exceeding the national
environmental standard of 25µg/m3.
Skin keratinocytes present the first barrier for environ-
mental pollutants, and it was shown that PM exposure could
upregulate pro-inflammatory mediators and AhR expression,
leading to increased ROS generation in keratinocytes (Choi
etal. 2011). Wei etal. (2017) demonstrated that organic
extracts containing PAHs with PM2.5 induced stronger oxi-
dative stress compared to those without PM2.5. It was also
shown that PM2.5 may penetrate the skin and have harmful
effects on viable skin cells, including keratinocytes (Krut-
mann etal. 2014; Li etal. 2017). A recent study demon-
strated that PM2.5 could increase ROS production and inhibit
the intracellular antioxidant system, which resulted in mor-
phological changes and decreased viability of keratinocytes
(Hu etal. 2017). Therefore, in the current study, we inves-
tigated the effects of oxidative stress induced by PM2.5 on
keratinocytes invitro and invivo. Our data indicate that
PM2.5 treatment promoted ROS generation (Fig.1a, c) and
caused structural damage, including DNA oxidation, lipid
peroxidation, and protein carbonylation (Fig.1f–k). Recent
studies have shown that ER stress is associated with oxida-
tive stress and that ROS may act as messengers between
these processes (Cao and Kaufman 2014; Laing etal. 2010).
Excessive ER Ca2+ release and mitochondrial Ca2+ overload
further amplify oxidative stress (Ly etal. 2017). Therefore,
we hypothesized that ROS overproduction induced by PM2.5
affected the ER which plays an important role in cellular
quality control and sensitivity to oxidative stress. Abnormal
ER stress is associated with autophagy-induced protein deg-
radation and activation of cytotoxic processes such as apop-
tosis (Schrock etal. 2013), which may be a key mechanism
underlying PM2.5 toxicity. GRP78 is a major ER chaperone
critical for protein quality control in the ER and activation of
ER transmembrane signaling molecules (Wang etal. 2009).
GRP78 interacts with misfolded proteins and promotes their
refolding, thereby playing an important role in regulating
three ER transmembrane proteins: PERK, IRE-1α, and acti-
vating transcription factor 6 (ATF6) (Mei etal. 2013). Our
data show that PM2.5 could induce IRE-1 phosphorylation,
upregulate GRP78 and CHOP expression, and activate the
ER stress pathway in human keratinocytes (Fig.2c, d). Fur-
thermore, ER stress is known to be strongly associated with
the disruption of cellular Ca2+ homeostasis, and our results
revealed that PM2.5-induced ER stress increased intracellular
Ca2+ levels, which was inhibited by NAC (Fig.2b).
Mitochondria are considered the main source of intra-
cellular ROS and mitochondrial dysfunction plays an
important role in the pathogenesis and/or progression
of various diseases. Our results demonstrate that PM2.5
induced structural alterations of mitochondria, including
swelling, which can deregulate the functional activity of
the mitochondrial respiratory chain and the production of
ROS, and lead to mitochondrial damage, suggesting that
a
b
Normal
1
PM2.5
2
PM2.5
3
PM2.5
4
Scale bar: 1 and 3=500 nm, 2 and 4=2 μm
Control PM2.5
1 2
2-1
Scale bar: 1 and 2=2 μm, 2-1=1 μm
Fig. 6 TEM analysis of HaCaT cell and mouse skin after PM2.5 treat-
ment. a Cells were treated with 50 µg/mL PM2.5 for 24h and ana-
lyzed for PM2.5 internalization (white arrow); 1 control, 2 internal-
ized PM2.5. Scale bars: 1 and 2, 2 µm; 21, 1µm. b Mouse skin
treated or not with 100µg/mL PM2.5 for 7 days. Compared to 1 nor-
mal untreated skin tissue, PM2.5-treated skin showed swelling of 2
mitochondria and 3 ER, and the presence of 4 autophagosomes. Scale
bars, 1 and 3, 500nm; 2 and 4, 2µm
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2089Archives of Toxicology (2018) 92:2077–2091
1 3
PM2.5 exposure promotes oxidative stress through destruc-
tion of mitochondria.
Autophagy is a regulated process of degradation and
recycling of dysfunctional organelles and proteins, which
are sequestered into autophagosomes that subsequently
fuse with lysosomes where the cargo is degraded by lyso-
somal hydrolases (Ryter etal. 2013); however, excessive
autophagy can directly cause cell death (Fulda and Kögel
2015). It has been reported that PM2.5-induced oxidative
stress could trigger autophagy in various cell types (Deng
etal. 2013, Su etal. 2017; Zhou etal. 2017). Consistent
with these findings, we observed stimulation of autophagy
in PM2.5-treated HaCaT keratinocytes invitro and mouse
keratinocytes invivo.
In conclusion, our study shows that PM2.5 causes skin
damage through induction of oxidative stress, which
results in the destruction of complex macromolecules and
cellular organelles, including the ER, mitochondria, and
lysosomes, and promotes apoptotic cell death (Fig.7).
Thus, our results contribute to understanding of the mech-
anisms underlying PM2.5-induced adverse effects on the
skin.
Acknowledgements This work was supported by grant from the Basic
Research Laboratory Program (NRF-2017R1A4A1014512) by the
National Research Foundation of Korea (NRF) Grant funded by the
Korea government (MSIP).
Compliance with ethical standards
Conflict of interest The authors declare no conflicts of interest.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creat iveco
mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
Air Korea site of Korea Environment Corporation (2015) Atmospheric
Environment Statistical Yearbook, Chap 2.2, Korea. http://www.
airko rea.or.kr/detai lView Down. Accessed 30 Oct 2017
Air Korea site of Korea Environment Corporation (2016) Atmospheric
Environment Statistical Yearbook, Chap 2.2, Korea. http://www.
airko rea.or.kr/detai lView Down. Accessed 30 Oct 2017
Air Korea site of Korea Environment Corporation (2017) Atmospheric
Environment Statistical Yearbook, Chap 3.1, Korea. http://www.
airko rea.or.kr/detai lView Down. Accessed 30 Oct 2017
Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D (2000)
Dynamic interaction of BiP and ER stress transducers in the
Fig. 7 PM2.5 causes skin injury
by increasing apoptosis through
oxidative stress and destruc-
tion of cellular organelles.
PM2.5-induced ROS generation
promotes ER stress, mito-
chondrial dysfunction, and
autophagy, leading to apoptotic
cell death and skin damage
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
2090 Archives of Toxicology (2018) 92:2077–2091
1 3
unfolded-protein response. Nat Cell Biol 2:326–332. https ://doi.
org/10.1038/35014 014
Cao SS, Kaufman RJ (2014) Endoplasmic reticulum stress and oxida-
tive stress in cell fate decision and human disease. Antioxid Redox
Signal 21:396–413. https ://doi.org/10.1089/ars.2014.5851
Chaudhary AK, Yadav N, Bhat TA, O’Malley J, Kumar S, Chandra D
(2016) A potential role of X-linked inhibitor of apoptosis protein
in mitochondrial membrane permeabilization and its implica-
tion in cancer therapy. Drug Discov Today 21:38–47. https ://doi.
org/10.1016/j.drudi s.2015.07.014
Choi H, Shin DW, Kim W, Doh SJ, Lee SH, Noh M (2011) Asian dust
storm particles induce a broad toxicological transcriptional pro-
gram in human epidermal keratinocytes. Toxicol Lett 200:92–99.
https ://doi.org/10.1016/j.toxle t.2010.10.019
Costa C, Catania S, De Pasquale R, Stancanelli R, Scribano GM, Mel-
chini A (2010) Exposure of human skin to benzo[a]pyrene: role
of CYP1A1 and aryl hydrocarbon receptor in oxidative stress
generation. Toxicology 271:83–86. https ://doi.org/10.1016/j.
tox.2010.02.014
Deng X, Zhang F, Rui W, Long F, Wang L, Feng Z, Chen D, Ding
W (2013) PM2.5-induced oxidative stress triggers autophagy in
human lung epithelial A549 cells. Toxicol InVitro 27:1762–1770.
https ://doi.org/10.1016/j.tiv.2013.05.004
Du Y, Xu X, Chu M, Guo Y, Wang J (2016) Air particulate matter and
cardiovascular disease: the epidemiological, biomedical and clini-
cal evidence. J Thorac Dis 8:E8-E19. https ://doi.org/10.3978/j.
issn.2072-1439.2015.11.37
Farah MA, Ali MA, Chen SM, Li Y, Al-Hemaid FM, Abou-Tarboush
FM, Al-Anazi KM, Lee J (2016) Silver nanoparticles synthesized
from Adenium obesum leaf extract induced DNA damage, apopto-
sis and autophagy via generation of reactive oxygen species. Col-
loids Surf B Biointerfaces 141:158–169. https ://doi.org/10.1016/j.
colsu rfb.2016.01.027
Fazeli G, Wehman AM (2017) Safely removing cell debris with LC3-
associated phagocytosis. Biol Cell 109:355–363. https ://doi.
org/10.1111/boc.20170 0028
Fritsche E, Schäfer C, Calles C, Bernsmann T, Bernshausen T, Wurm
M, Hübenthal U, Cline JE, Hajimiragha H, Schroeder P, Klotz
LO, Rannug A, Fürst P, Hanenberg H, Abel J, Krutmann J (2007)
Lightening up the UV response by identification of the arylhy-
drocarbon receptor as a cytoplasmatic target for ultraviolet B
radiation. Proc Natl Acad Sci USA 104:8851–8856. https ://doi.
org/10.1073/pnas.07017 64104
Fulda S, Kögel D (2015) Cell death by autophagy: emerging molecu-
lar mechanisms and implications for cancer therapy. Oncogene
34:5105–5113. https ://doi.org/10.1038/onc.2014.458
Gualtieri M, Longhin E, Mattioli M, Mantecca P, Tinaglia V, Man-
gano E, Proverbio MC, Bestetti G, Camatini M, Battaglia C
(2012) Gene expression profiling of A549 cells exposed to Milan
PM2.5. Toxicol Lett 209:136–145. https ://doi.org/10.1016/j.toxle
t.2011.11.015
Guo Z, Hong Z, Dong W, Deng C, Zhao R, Xu J, Zhuang G, Zhang R
(2017) PM2.5-induced oxidative stress and mitochondrial dam-
age in the nasal mucosa of rats. Int J Environ Res Public Health
14:E134. https ://doi.org/10.3390/ijerp h1402 0134
Han X, Liang WL, Zhang Y, Sun LD, Liang WY (2016) Effect of
atmospheric fine particles on epidermal growth factor receptor
mRNA expression in mouse skin tissue. Genet Mol Res. https ://
doi.org/10.4238/gmr.15017 188
Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflam-
matory basis of metabolic disease. Cell 140:900–917. https ://doi.
org/10.1016/j.cell.2010.02.034
Hu R, Xie XY, Xu SK, Wang YN, Jiang M, Wen LR, Lai W, Guan L
(2017) PM2.5 exposure elicits oxidative stress responses and mito-
chondrial apoptosis pathway activation in HaCaT keratinocytes.
Chin Med J 130:2205–2214. https ://doi.org/10.4103/0366-
6999.21294 2
Jakobsen CH, Størvold GL, Bremseth H, Follestad T, Sand K, Mack
M, Olsen KS, Lundemo AG, Iversen JG, Krokan HE, Schønberg
SA (2008) DHA induces ER stress and growth arrest in human
colon cancer cells: associations with cholesterol and calcium
homeostasis. J Lipid Res 49:2089–2100. https ://doi.org/10.1194/
jlr.M7003 89-JLR20 0
Jeong JW, Cha HJ, Han MH, Hwang SJ, Lee DS, Yoo JS, Choi IW,
Kim S, Kim HS, Kim GY, Hong SH, Park C, Lee HJ, Choi YH
(2017) Spermidine protects against oxidative stress in inflamma-
tion models using macrophages and zebrafish. Biomol Ther. https
://doi.org/10.4062/biomo lther .2016.272
Jung S, Lim J, Kwon S, Jeon S, Kim J, Lee J, Kim S (2017) Charac-
terization of particulate matter from diesel passenger cars tested
on chassis dynamometers. J Environ Sci 54:21–32. https ://doi.
org/10.1016/j.jes.2016.01.035
Jux B, Kadow S, Luecke S, Rannug A, Krutmann J, Esser C (2011)
The aryl hydrocarbon receptor mediates UVB radiation-induced
skin tanning. J Investig Dermatol 131:203–210. https ://doi.
org/10.1038/jid.2010.269
Kaneto H, Matsuoka TA, Nakatani Y, Kawamori D, Miyatsuka T, Mat-
suhisa M, Yamasaki Y (2005) Oxidative stress, ER stress, and the
JNK pathway in type 2 diabetes. J Mol Med 83:429–439. https ://
doi.org/10.1007/s0010 9-005-0640-x
Kim HB, Yoo BS (2016) Propolis inhibits UVA-induced apoptosis of
human keratinocyte HaCaT cells by scavenging ROS. Toxicol Res
32:345–351. https ://doi.org/10.5487/TR.2016.32.4.345
Kim KE, Cho D, Park HJ (2016) Air pollution and skin diseases:
adverse effects of airborne particulate matter on various skin
diseases. Life Sci 152:126–134. https ://doi.org/10.1016/j.
lfs.2016.03.039
Kouassi KS, Billet S, Garçon G, Verdin A, Diouf A, Cazier F, Djaman
J, Courcot D, Shirali P (2010) Oxidative damage induced in A549
cells by physically and chemically characterized air particulate
matter (PM2.5) collected in Abidjan, Côte d’Ivoire. J Appl Toxi-
col 30:310–320. https ://doi.org/10.1002/jat.1496
Krutmann J, Liu W, Li L, Pan X, Crawford M, Sore G, Seite S (2014)
Pollution and skin: from epidemiological and mechanistic studies
to clinical implications. J Dermatol Sci 76:163–168. https ://doi.
org/10.1016/j.jderm sci.2014.08.008
Laing S, Wang G, Briazova T, Zhang C, Wang A, Zheng Z, Gow A,
Chen AF, Rajagopalan S, Chen LC, Sun Q, Zhang K (2010) Air-
borne particulate matter selectively activates endoplasmic reticu-
lum stress response in the lung and liver tissues. Am J Physiol Cell
Physiol 299:C736–C749. https ://doi.org/10.1152/ajpce ll.00529
.2009
Lee BK, Smith TJ, Garshick E, Natkin J, Reaser P, Lane K, Lee HK
(2005) Exposure of trucking company workers to particulate mat-
ter during the winter. Chemosphere 61:1677–1690. https ://doi.
org/10.1016/j.chemo spher e.2005.03.091
Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxida-
tive stress: cross-talk and redox signalling. Biochem J 441:523–
540. https ://doi.org/10.1042/BJ201 11451
Lee CW, Lin ZC, Hu SC, Chiang YC, Hsu LF, Lin YC, Lee IT, Tsai
MH, Fang JY (2016) Urban particulate matter down-regulates
filaggrin via COX2 expression/PGE2 production leading to skin
barrier dysfunction. Sci Rep 6:27995. https ://doi.org/10.1038/
srep2 7995
Lee YK, Kim SW, Park JY, Kang WC, Kang YJ, Khang D (2017)
Suppression of human arthritis synovial fibroblasts inflammation
using dexamethasone-carbon nanotubes via increasing caveolin-
dependent endocytosis and recovering mitochondrial membrane
potential. Int J Nanomed 12:5761–5779. https ://doi.org/10.2147/
IJN.S1421 22
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
2091Archives of Toxicology (2018) 92:2077–2091
1 3
Li D, Li L, Li P, Li Y, Chen X (2015) Apoptosis of HeLa cells induced
by a new targeting photosensitizer-based PDT via a mitochondrial
pathway and ER stress. Onco Targets Ther 8:703–711. https ://doi.
org/10.2147/OTT.S7637 0
Li Q, Kang Z, Jiang S, Zhao J, Yan S, Xu F, Xu J (2017) Effects of
ambient fine particles PM2.5 on human HaCaT cells. Int J Envi-
ron Res Public Health 14:E72. https ://doi.org/10.3390/ijerp h1401
0072
Liu Q, Xu C, Ji GX, Liu H, Shao WT, Zhang CL, Gu A, Zhao P (2017)
Effect of exposure to ambient PM2.5 pollution on the risk of res-
piratory tract diseases: a meta-analysis of cohort studies. J Biomed
Res 31:130–142. https ://doi.org/10.7555/JBR.31.20160 071
Ly LD, Xu S, Choi SK, Ha CM, Thoudam T, Cha SK, Wiederkehr
A, Wollheim CB, Lee IK, Park KS (2017) Oxidative stress and
calcium dysregulation by palmitate in type 2 diabetes. Exp Mol
Med 49:e291. https ://doi.org/10.1038/emm.2016.157
Mei Y, Thompson MD, Cohen RA, Tong X (2013) Endoplasmic
reticulum stress and related pathological processes. J Pharmacol
Biomed Anal 1:1000107
Morita M, Naito Y, Yoshikawa T, Niki E (2016) Plasma lipid oxi-
dation induced by peroxynitrite, hypochlorite, lipoxygenase and
peroxyl radicals and its inhibition by antioxidants as assessed by
diphenyl-1-pyrenylphosphine. Redox Biol 8:127–135. https ://doi.
org/10.1016/j.redox .2016.01.005
Nishitoh H (2012) CHOP is a multifunctional transcription factor
in the ER stress response. J Biochem 151:217–219. https ://doi.
org/10.1093/jb/mvr14 3
Park JE, Piao MJ, Kang KA, Shilnikova K, Hyun YJ, Oh SK, Jeong
YJ, Chae S, Hyun JW (2017) A Benzylideneacetophenone deriva-
tive induces apoptosis of radiation-resistant human breast cancer
cells via oxidative stress. Biomol Ther 25:404–410. https ://doi.
org/10.4062/biomo lther .2017.010
Piao MJ, Kim KC, Choi JY, Choi J, Hyun JW (2011) Silver nano-
particles down-regulate Nrf2-mediated 8-oxoguanine DNA gly-
cosylase 1 through inactivation of extracellular regulated kinase
and protein kinase B in human Chang liver cells. Toxicol Lett
207:143–148. https ://doi.org/10.1016/j.toxle t.2011.09.002
Ryter SW, Cloonan SM, Choi AM (2013) Autophagy: a critical regula-
tor of cellular metabolism and homeostasis. Mol Cells 36:7–16.
https ://doi.org/10.1007/s1005 9-013-0140-8
Schrock JM, Spino CM, Longen CG, Stabler SM, Marino JC, Pasternak
GW, Kim FJ (2013) Sequential cytoprotective responses to sigma1
ligand-induced endoplasmic reticulum stress. Mol Pharmacol
84:751–762. https ://doi.org/10.1124/mol.113.08780 9
Soeur J, Belaïdi JP, Chollet C, Denat L, Dimitrov A, Jones C, Perez P,
Zanini M, Zobiri O, Mezzache S, Erdmann D, Lereaux G, Eilstein
J, Marrot L (2017) Photo-pollution stress in skin: traces of pol-
lutants (PAH and particulate matter) impair redox homeostasis in
keratinocytes exposed to UVA1. J Dermatol Sci 86:162–169. https
://doi.org/10.1016/j.jderm sci.2017.01.007
Song S, Lee K, Lee YM, Lee JH, Lee SI, Yu SD, Paek D (2011) Acute
health effects of urban fine and ultrafine particles on children
with atopic dermatitis. Environ Res 111:394–399. https ://doi.
org/10.1016/j.envre s.2010.10.010
Su R, Jin X, Zhang W, Li Z, Liu X, Ren J (2017) Particulate mat-
ter exposure induces the autophagy of macrophages via oxida-
tive stress-mediated PI3K/AKT/mTOR pathway. Chemosphere
167:444–453. https ://doi.org/10.1016/j.chemo spher e.2016.10.024
Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lyso-
somal turnover, but not a cellular level, of endogenous LC3 is a
marker for autophagy. Autophagy 1:84–91
Vogel CF, Chang WL, Kado S, McCulloh K, Vogel H, Wu D, Haar-
mann-Stemmann T, Yang G, Leung PS, Matsumura F, Gershwin
ME (2016) Transgenic overexpression of aryl hydrocarbon recep-
tor repressor (AhRR) and AhR-mediated induction of CYP1A1,
cytokines, and acute toxicity. Environ Health Perspect 124:1071–
1083. https ://doi.org/10.1289/ehp.15101 94
Wang M, Wey S, Zhang Y, Ye R, Lee AS (2009) Role of the unfolded
protein response regulator GRP78/BiP in development, cancer,
and neurological disorders. Antioxid Redox Signal 11:2307–2316.
https ://doi.org/10.1089/ARS.2009.2485
Wang Z, Liu D, Varin A, Nicolas V, Courilleau D, Mateo P, Caubere C,
Rouet P, Gomez AM, Vandecasteele G, Fischmeister R, Brenner
C (2016) A cardiac mitochondrial cAMP signaling pathway regu-
lates calcium accumulation, permeability transition and cell death.
Cell Death Dis 7:e2198. https ://doi.org/10.1038/cddis .2016.106
Wang Y, Xiong L, Tang M (2017) Toxicity of inhaled particulate mat-
ter on the central nervous system: neuroinflammation, neuropsy-
chological effects and neurodegenerative disease. J Appl Toxicol
37:644–667. https ://doi.org/10.1002/jat.3451
Wei H, Feng Y, Liang F, Cheng W, Wu X, Zhou R, Wang Y (2017)
Role of oxidative stress and DNA hydroxymethylation in the neu-
rotoxicity of fine particulate matter. Toxicology 380:94–103. https
://doi.org/10.1016/j.tox.2017.01.017
Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress:
cell life and death decisions. J Clin Investig 115:2656–2664. https
://doi.org/10.1172/JCI26 373
Yao J, Jiao R, Liu C, Zhang Y, Yu W, Lu Y, Tan R (2016) Assess-
ment of the cytotoxic and apoptotic effects of chaetominine in a
human leukemia cell line. Biomol Ther 24:147–155. https ://doi.
org/10.4062/biomo lther .2015.093
Zhao J, Gao Z, Tian Z, Xie Y, Xin F, Jiang R, Kan H, Song W (2013)
The biological effects of individual-level PM(2.5) exposure on
systemic immunity and inflammatory response in traffic police-
men. Occup Environ Med 70:426–431. https ://doi.org/10.1136/
oemed -2012-10086 4
Zhou ZX, Shao T, Qin MN, Miao XY, Chang Y, Sheng W, Wu FS,
Yu YJ (2017) The effects of autophagy on vascular endothe-
lial cells induced by airborne PM2.5. J Environ Sci. https ://doi.
org/10.1016/j.jes.2017.05.019
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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... Through inhalation, ingestion, and skin contact, it has led to the accumulation of particulate matter (PM) in various organs throughout the body (1). Emissions of particulate matter from fossil fuelsparticularly dieselare a major source of air pollution (2). The toxicological evaluation of fine (size < 2.5 mm) particulate matter (PM 2.5 ) has been conducted in the epidermal (2), respiratory (3), immune (4), nervous (5), and cardiovascular systems (6). ...
... Diesel particulate matter (NIST ® SRM ® 1650b, Sigma-Aldrich, St. Louis, MO, USA) was diluted in dimethyl sulfoxide (DMSO) (10 mg/mL) to form a stock solution and was stored at -20°C. Before use, the stock solution was sonicated for 30 min to prevent particle aggregation (2). The stock solution was then diluted in a Claycomb medium (Sigma-Aldrich) for treatment. ...
Article
Full-text available
Particulate matter (PM) in polluted air can be exposed to the human body through inhalation, ingestion, and skin contact, accumulating in various organs throughout the body. Organ accumulation of PM is a growing health concern, particularly in the cardiovascular system. PM emissions are formed in the air by solid particles, liquid droplets, and fuel – particularly diesel – combustion. PM 2.5 (size < 2.5 μm particle) is a major risk factor for approximately 200,000 premature deaths annually caused by air pollution. This study assessed the deleterious effects of diesel-derived PM 2.5 exposure in HL-1 mouse cardiomyocyte cell lines. The PM 2.5 -induced biological changes, including ultrastructure, intracellular reactive oxygen species (ROS) generation, viability, and intracellular ATP levels, were analyzed. Moreover, we analyzed changes in transcriptomics using RNA sequencing and metabolomics using gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in PM 2.5 -treated HL-1 cells. Ultrastructural analysis using transmission electron microscopy revealed disruption of mitochondrial cristae structures in a PM 2.5 dose-dependent manner. The elevation of ROS levels and reduction in cell viability and ATP levels were similarly observed in a PM 2.5 dose-dependently. In addition, 6,005 genes were differentially expressed (fold change cut-off ± 4) from a total of 45,777 identified genes, and 20 amino acids (AAs) were differentially expressed (fold change cut-off ± 1.2) from a total of 28 identified AAs profiles. Using bioinformatic analysis with ingenuity pathway analysis (IPA) software, we found that the changes in the transcriptome and metabolome are highly related to changes in biological functions, including homeostasis of Ca ²⁺ , depolarization of mitochondria, the function of mitochondria, synthesis of ATP, and cardiomyopathy. Moreover, an integrated single omics network was constructed by combining the transcriptome and the metabolome. In silico prediction analysis with IPA predicted that upregulation of mitochondria depolarization, ROS generation, cardiomyopathy, suppression of Ca ²⁺ homeostasis, mitochondrial function, and ATP synthesis occurred in PM 2.5 -treated HL-1 cells. In particular, the cardiac movement of HL-1 was significantly reduced after PM 2.5 treatment. In conclusion, our results assessed the harmful effects of PM 2.5 on mitochondrial function and analyzed the biological changes related to cardiac movement, which is potentially associated with cardiovascular diseases.
... The size, composition, and the origin of these particles are based on their microenvironment [1]. PM contains organic compounds that can readily penetrate the skin [2]. The skin is the largest organ in the human body and provides the largest interface between the body and external environment. ...
Article
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The increasing airborne particulate matter (PM) consisting of environmental contaminants such as dust, aerosols, and fibers has become a global concern by causing oxidative stress that leads to apoptosis and skin damage. The current study evaluated the protective effect of Caulerpa racemosa (CR) against PM-induced skin damage using human keratinocytes and a zebrafish model. The clionasterol-rich hexane fraction (CRHF2) of CR exhibited superior protective activity through downregulating intracellular reactive oxygen species levels and mitochondrial ROS levels, as well as the PM-induced increase in apoptotic body formation and upregulation of apoptotic signaling pathway proteins, along with sub-G1 cell accumulation dose-dependently. Furthermore, in vivo results showed that CRHF2 potentially downregulates PM-induced cell death, ROS, and NO production in the zebrafish model. Hence, the results evidenced that the protective effect of CRHF2 is caused by inhibiting oxidative stress and mitochondrial-mediated apoptosis in cells. Therefore, C. racemosa has the potential to be used in the development of pharmaceuticals to attenuate PM-induced skin diseases.
... 44 In a particulate matter induced skin damage model, ginsenosides showed excellent skin protection and could inhibit mitochondria and ER stress-dependent apoptosis. 45 Most probably phenolic compounds like the flavonoid glycosylates and the saponins present in BFKE contributes to the activity of the extract. ...
Article
Full-text available
Katharina Kappler,1 Torsten Grothe,1 Shalini Srivastava,2 Manjiri Jagtap3 1Mibelle Group Biochemistry, Buchs, CH-5033, Switzerland; 2Vedic Lifesciences Private Limited, Mumbai, India; 3Skin Cure N Care, Mumbai, IndiaCorrespondence: Torsten Grothe, Email torsten.grothe@mibellegroup.comBackground: The skin is primarily affected by aging, especially when it is exposed to particulate matter present in the environment. It has been hypothesized that consumption of products with known antioxidant properties would help combat factors associated with both intrinsic and extrinsic aging factors.Objective: The aim of the present study was to evaluate the effect of the formulation Blue Fenugreek Kale Extract (BFKE) on skin aging.Methods: In this study, the effect of BFKE on protein oxidation was determined in human dermal fibroblasts by analysis of the level of protein carbonylation after cells were stressed with either H2O2 or urban pollution consisting of particulate matter and UV-A. Furthermore, a randomized, double-blind, placebo-controlled clinical study that evaluated the effect of BFKE consumption over a period of 56 days in 59 volunteers was performed. The major parameter studied was skin barrier dysfunction through the assessment of Transepidermal Water Loss (TEWL). Additional parameters analyzed clinically include skin moisture content, participant self-assessment of skin parameters, wrinkle severity, skin sagging and elasticity. Furthermore, low grade and allergic inflammatory biomarker levels were measured at the start and end of the treatment period, along with oxidative stress assessment using blood malondialdehyde levels.Results: BFKE significantly reduced protein carbonylation in human dermal fibroblasts stressed with urban pollution. In the clinical study, the TEWL level reduced significantly and at the same time the skin moisture content levels increased by end of the treatment period. No significant changes were observed in wrinkle severity, skin sagging, elasticity, inflammatory and oxidative stress biomarker levels. Participant and investigator perception of treatment was significantly greater after product consumption, as was the improvement in skin parameters based on participant self-assessment.Conclusion: BFKE reduces protein oxidation induced by H2O2 and restores skin barrier function and skin hydration, while also combating early signs of aging.Keywords: blue fenugreek kale extract, transepidermal water loss, skin barrier function, skin aging, antioxidant, pollution
... Exposure of HaCaT cells to ambient particles containing benzopyrenes led to excessive oxidative stress, which could promote mitochondrial swelling, deregulation of the respiratory chain, and further production of reactive oxygen species [41]. The disruption of mitochondria l membrane potential plays an important role in the induction and progression of various skin of skin diseases including skin cancer [3]. ...
Article
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds derived mostly from the incomplete combustion of fossil fuels and biomass. Human skin can absorb PAHs and the uptake increases with their molar mass and lipophilicity. Benzopyrene is high-molecular-weight PAH frequently appearing in ambient pollution. It exists in two isomeric forms: benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP), which exhibit different biological activity. Although certain properties of benzopyrenes suggested photoreactivity of the compounds, no direct measurements were previously conducted to characterize their photochemical activity. In this study, quantum yield and action spectra of singlet oxygen photogeneration by BaP and BeP were measured by time-resolved near-infrared phosphorescence and the ability of both compounds to photogenerate superoxide anion was assessed by electron paramagnetic resonance (EPR) spin-trapping. The measurements revealed high efficiency of benzopyrenes to photogenerate singlet oxygen and their ability to photogenerate superoxide anion. Using HaCaT cells as single-layer skin model, we demonstrated concentration-dependent and light-dependent cytotoxicity of BaP and BeP. The compounds induced damage to the cell mitochondria and elevated the levels of intracellular reactive oxygen species.
... The generation of reactive oxygen species (ROS) is an important mechanism for PM2.5-induced skin damage. When HaCaT cells (immortalized nontumorigenic human keratinocytes) were exposed to PM2.5, intracellular ROS were increased, and subsequently, the increased ROS induced endoplasmic reticulum stress and mitochondrial damage (Piao et al., 2018). Dong et al. also reported that treatment of HaCaT cells with PM2.5 increased intracellular ROS levels and subsequent upregulation of NLRP1 and IL-1β via NF-κB activation (Dong et al., 2020). ...
Article
Skin is the primary tissue exposed to ambient air pollution because it acts as an interface between the body and the surrounding atmosphere. We previously reported that particulate matter 2.5 (PM2.5) induced oxidative stress and subsequent chemokine release in the human epidermis, followed by neutrophil chemotaxis. We identified in this study that the leaf extracts from Camellia sinensis and Argania spinosa showed high radical scavenging activity as evaluated by 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazinyl and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid assays. PM2.5 exposure induced lipid peroxidation, IL-8 release and neutrophil migration in human 3-dimensional cultured epidermis. Pretreatment with leaf extracts from Camellia sinensis or Argania spinosa significantly suppressed the above harmful effects elicited by PM2.5. Taken together, both extracts can protect the epidermis from PM2.5 exposure. Camellia sinensis and Argania spinosa extracts could be added to a novel cosmetic that protects skin from air pollution.
... Cells were cultured under high glucose in a chamber slide at 1.5 × 10 5 cells/mL. The 8−oxoG modification was observed under a confocal microscope using the avidin−TRITC conjugate [23]. ...
Article
Full-text available
Neurodegenerative diseases are associated with neuronal cell death through apoptosis. Apoptosis is tightly associated with the overproduction of reactive oxygen species (ROS), and high glucose levels contribute to higher oxidative stress in diabetic patients. Hesperidin, a natural active compound, has been reported to scavenge free radicals. Only a few studies have explored the protective effects of hesperidin against high glucose−induced apoptosis in SH−SY5Y neuronal cells. Glucose stimulated neuronal cells to generate excessive ROS and caused DNA damage. In addition, glucose triggered endoplasmic reticulum stress and upregulated cytoplasmic as well as mitochondrial calcium levels. Hesperidin inhibited glucose−induced ROS production and mitigated the associated DNA damage and endoplasmic reticulum stress. The downregulation of antiapoptotic protein Bcl−2 following glucose treatment was reversed by a hesperidin treatment. Furthermore, hesperidin repressed the glucose−induced Bcl−2−associated X protein, cleaved caspase−9, and cleaved caspase−3. Hesperidin also suppressed the glucose−induced phosphorylation of extracellular signal−regulated kinase and c−Jun N−terminal kinase. The current results confirmed that hesperidin could protect neuronal cells against glucose−induced ROS. Mechanistically, hesperidin was shown to promote cell viability via attenuation of the mitogen−activated protein kinase signaling pathway.
... Since they are natural ligands of the aryl hydrocarbon receptor (AhR), they usually disturb cell differentiation and lipogenesis. AhR signaling mediates cell apoptosis, oxidative stress, hyperpigmentation, and subcellular organelle dysfunction induced by particulate matter (PM) 2.5 in HaCaT keratinocytes (Piao et al., 2018;Shi et al., 2021). Correspondingly, Liu et al. have shown that a standard reference material of air pollution PM induced human skin keratinocyte and dermal fibroblast aging through cell growth inhibition and cell arrest, which could cause skin barrier damage and collagen degradation. ...
Article
Full-text available
Sebaceous glands (SGs) originate from hair follicular stem cells and secrete lipids to lubricate the skin. The coordinated effects of intrinsic and extrinsic aging factors generate degradation of SGs at a late age. Senescence of SGs could be a mirror of the late aging of both the human body and skin. The procedure of SG aging goes over an initial SG hyperplasia at light-exposed skin areas to end with SG atrophy, decreased sebum secretion, and altered sebum composition, which is related to skin dryness, lack of brightness, xerosis, roughness, desquamation, and pruritus. During differentiation and aging of SGs, many signaling pathways, such as Wnt/β-catenin, c-Myc, aryl hydrocarbon receptor (AhR), and p53 pathways, are involved. Random processes lead to random cell and DNA damage due to the production of free radicals during the lifespan and neuroendocrine system alterations. Extrinsic factors include sunlight exposure (photoaging), environmental pollution, and cigarette smoking, which can directly activate signaling pathways, such as Wnt/β-catenin, Notch, AhR, and p53 pathways, and are probably associated with the de-differentiation and hyperplasia of SGs, or indirectly activate the abovementioned signaling pathways by elevating the inflammation level. The production of ROS during intrinsic SG aging is less, the signaling pathways are activated slowly and mildly, and sebocytes are still differentiated, yet terminal differentiation is not completed. With extrinsic factors, relevant signaling pathways are activated rapidly and fiercely, thus inhibiting the differentiation of progenitor sebocytes and even inducing the differentiation of progenitor sebocytes into keratinocytes. The management of SG aging is also mentioned.
... Particles can directly interact and eventually penetrate cutaneous tissue, contributing to increased oxidative stress, activation of inflammatory pathways, DNA damage and skin aging [5,63,64]. Indeed, despite the inability of O 3 to cross the skin barrier, some PM components, such as polyaromatic carbons (PAHs), can penetrate transdermally or through hair follicles, triggering the production of ROS and lipid peroxidation (4-HNE), leading to apoptosis, DNA and mitochondria damage, activation of pro-inflammatory pathways (NF-kβ, AP1) and the antioxidant response (NRF2) [5,[63][64][65][66]. In addition, oxides of nitrogen display an oxidizing effect in skin tissue [67], and their effect on human skin is strictly related to ultrafine PM and black carbon, since they are all emitted during traffic and industries emission [68,69]. ...
Article
Full-text available
Our current understanding of the pathogenesis of skin aging includes the role of ultraviolet light, visible light, infrared, pollution, cigarette smoke and other environmental exposures. The mechanism of action common to these exposures is the disruption of the cellular redox balance by the directly or indirectly increased formation of reactive oxygen species that overwhelm the intrinsic antioxidant defense system, resulting in an oxidative stress condition. Altered redox homeostasis triggers downstream pathways that contribute to tissue oxinflammation (cross-talk between inflammation and altered redox status) and accelerate skin aging. In addition, both ultraviolet light and pollution increase intracellular free iron that catalyzes reactive oxygen species generation via the Fenton reaction. This disruption of iron homeostasis within the cell further promotes oxidative stress and contributes to extrinsic skin aging. More recent studies have demonstrated that iron chelators can be used topically and can enhance the benefits of topically applied antioxidants. Thus, an updated, more comprehensive approach to environmental or atmospheric aging protection should include sun protective measures, broad spectrum sunscreens, antioxidants, chelating agents, and DNA repair enzymes.
Article
Urban particulate matter (PM) is a major air pollutant that triggers molecular processes and is detrimental to the skin. We investigated the protective effects of tocotrienol-rich fraction (TRF) against urban PM-induced skin ageing, inflammation, and skin barrier dysfunction in human epidermal keratinocytes. Alpha-tocopherol (αTP) and retinoic acid (RA) were used as comparators. Our results showed that TRF significantly restored cell viability and alleviated increased intracellular reactive oxygen radicals in PM-treated keratinocytes. In addition, TRF significantly downregulated the activation of mitogen-activated protein kinases in PM-stimulated keratinocytes. This was substantiated by lower protein expression in the phosphorylation of extracellular signal-regulated kinase, Jun N-terminal kinase, and p38. This resulted in the inhibition of cyclooxygenase-2 expression, which is a downstream inflammatory mediator. TRF significantly protected skin barrier function upon exposure to PM by upregulating filaggrin, transglutaminase-1, and involucrin. In contrast, αTP and RA did not exhibit protective effects against skin damages in the PM-treated keratinocytes. Overall, this study suggests that TRF possesses antioxidant, anti-inflammatory, and skin barrier protective properties, and may serve as a potential ingredient in personal care and cosmeceutical industries to combat skin damage due to air pollution.
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Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca²⁺ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca²⁺ release and thereby deplete ER Ca²⁺ stores. The resulting ER Ca²⁺ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca²⁺ release, cytosolic and mitochondrial matrix Ca²⁺ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca²⁺ depletion mediate persistent Ca²⁺ influx, further impairing cytosolic and mitochondrial Ca²⁺ homeostasis. Mitochondrial Ca²⁺ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.
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The purpose of this study was to examine the direct toxicity of PM2.5 collected from Beijing on human umbilical vein endothelial cells (HUVEC). A Cell Counting Kit 8 (CCK8) assay demonstrated that PM2.5 exposure decreased the proliferation of HUVECs in a dose-dependent manner. We also found that PM2.5 exposure induced autophagy in HUVECs, as evidenced by: (1) an increased number of double-membrane vesicles; (2) enhanced conversion and punctuation of the microtubule-associated protein light chain 3 (LC3); and (3) decreased levels of the selective autophagy substrate p62 in a time-dependent manner. Furthermore, promoting autophagy in PM2.5-exposed HUVECs with rapamycin increased the cell survival rate, whereas inhibiting autophagy via 3-methyladenine significantly decreased cell survival. These results demonstrate that PM2.5 exposure can induce cytotoxicity and autophagy in HUVECs and that autophagy play a protective role against PM2.5-induced cytotoxicity. The findings of the present study imply a direct toxic effect of PM2.5 on HUVECs and provide novel insight into the mechanism of cardiovascular diseases caused by PM2.5 exposure.
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The International Agency for Research on Cancer and the World Health Organization have designated airborne particulates, including particulates of median aerodynamic diameter ≤ 2.5 μm (PM2.5), as Group 1 carcinogens. It has not been determined, however, whether exposure to ambient PM2.5 is associated with an increase in respiratory related diseases. This meta-analysis assessed the association between exposure to ambient fine particulate matter (PM2.5) and the risk of respiratory tract diseases, using relevant articles extracted from PubMed,Web of Science, and Embase. In results, of the 1,126 articles originally identified, 35 (3.1%) were included in this meta-analysis. PM2.5 was found to be associated with respiratory tract diseases. After subdivision by age group, respiratory tract disease, and continent, PM2.5 was strongly associated with respiratory tract diseases in children, in persons with cough, lower respiratory illness, and wheezing, and in individuals from North America, Europe, and Asia. The risk of respiratory tract diseases was greater for exposure to traffic-related than non-traffic-related air pollution. In children, the pooled relative risk (RR) represented significant increases in wheezing (8.2%), cough (7.5%), and lower respiratory illness (15.3%). The pooled RRs in children were 1.091 (95%CI: 1.049, 1.135) for exposure to < 25 μg/m³ PM2.5, and 1.126 (95%CI: 1.067, 1.190) for exposure to ≥ 25 μg/m³ PM2.5. In conclusion, exposure to ambient PM2.5 was significantly associated with the development of respiratory tract diseases, especially in children exposed to high concentrations of PM2.5.
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