Chemosphere 224 (2019) 29e38

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Exposure to ambient fine particles causes abnormal energy

metabolism and ATP decrease in lung tissues
Xiaoting Jin a, Huilan Su a, Guobin Ding b, Zhendong Sun c, Zhuoyu Li a, b, d, *
a
Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China
b
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan
030006, China
c
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
Beijing 100085, China
d
School of Life Science, Shanxi University, Taiyuan 030006, China

h i g h l i g h t s

Haze dosage PM2.5 exposure led to the occurrence of lung tissue damage and reduction of ATP contents.

Winter PM2.5-reduced ATP production was slightly notable than that of summer PM2.5.

The activity of TCA cycle in lung tissues was weakened by seasonal PM2.5 exposure.

The level of glycolysis in lung tissues was strengthened in response to seasonal PM2.5.

a r t i c l e i n f o a b s t r a c t

Article history: Airborne fine particles, generating from human activities, have drawn increasing attention due to their
Received 6 November 2018 potential lung health hazards. The currently available toxicological data on fine particles is still not
Received in revised form sufficient to explain their cause-and-effect. Based on well reported critical role of ATP on maintaining
15 January 2019
lung structure and function, the alterations of ATP production and energy metabolism in lungs of rats
Accepted 17 February 2019
Available online 18 February 2019
exposed to different dosages of seasonal PM2.5 were investigated. Haze dosage PM2.5 exposure was
demonstrated to reduce the ATP production. Activity of critical enzymes in TCA cycle, such as malate
Handling Editor: A. Gies dehydrogenase (MDH) and citrate synthase (CS), and expression of mitochondrial respiration chain genes
were attenuated in groups exposed to haze dosage PM2.5. In contrast, there was prominent augment of
Keywords: glycolytic markers at haze dosage PM2.5, including metabolite contents (pyruvate and lactic acid),
PM2.5 enzyme activities (hexokinase (HK) and pyruvate kinase (PKM)), along with mRNA levels of PKM and
ATP LDH. Consequently, sub-chronic exposure to seasonal haze PM2.5 caused reduction in ATP generation and
TCA cycle metabolic rewiring from TCA cycle to glycolysis. Our findings can help better understand the toxico-
Glycolysis
logical mechanism of lung disease caused by particulate air pollution.
Metabolic rewiring
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction
Fine particulate matter <2.5 mm (PM2.5) can penetrate into the
Abbreviations: PM2.5, fine particulate matter; ATP, adenosine triphosphate;
QFFs, quartz fiber filters; SEM, scanning electron microscopy; TCA cycle, tricar-
boxylic acid cycle; MDA, malondialdehyde; CS, citrate synthase; MDH, malate de-
hydrogenase; Glut1, glucose transporter 1; HK, hexokinase; PKM, pyruvate kinase;
LDH, lactate dehydrogenase; qRT-PCR, quantitative real time PCR.
* Corresponding author. Institutes of Biomedical Sciences, Shanxi University,
Taiyuan 030006, China.
E-mail address: lzy@sxu.edu.cn (Z. Li).
lungs and alveoli (or alveolar sacs) areas easily (Meng and Zhang,
2006). Therefore, the organ is recognized as the most vulnerable
tissue for PM2.5 exposure. Epidemiologic and in vivo studies using
animals have shown that PM2.5 exposure was a risk factor for the
changes in function and/or histology of lung, respiratory diseases
and lung cancer (Brunekreef and Holgate, 2002; Wu et al., 2013;
Senthilkumar et al., 2014). Thus, it is of importance to investigate
the in-depth mechanism of PM2.5-induced lung disease for a better
understanding of PM2.5 toxicity and mode of action.
Poor lung function is a vital predictor of mortality in patients
with lung disease and general population (Sharma and Goodwin,

https://doi.org/10.1016/j.chemosphere.2019.02.116
0045-6535/© 2019 Elsevier Ltd. All rights reserved.
30 X. Jin et al. / Chemosphere 224 (2019) 29e38
2006). Therefore, lung function test has been extensively used in
PM2.5-induced lung toxicity. Many studies have revealed that PM2.5
was associated with the reduced lung function due to its short-term
or long-term exposure (Gehring et al., 2013; Gotschi et al., 2008).
Many researches indicate that the damaging mechanisms of PM2.5
on the respiratory system may include contribution from free
radical peroxidation, imbalanced intracellular calcium homeostasis
and inflammatory injury (Xing et al., 2016). Nevertheless, the un-
derlying mechanism still remains elusive. As an important respi-
ratory organ, the lung needs high consumption of adenosine
triphosphate (ATP), and the production of ATP is involved in a va-
riety of fundamental cellular biological processes. The cellular en-
ergy metabolism determines the lung function under homeostatic
and stress conditions (Pierdominici et al., 2014; Xu et al., 2014).
Moreover, several xenobiotics, including various drugs and envi-
ronmental pollutants or toxicants, have been recently reported to
change the ATP production, thereby altering cell physiology (Xu
et al., 2014; Grattagliano et al., 2009). However, the knowledge
on the energy alteration due to PM2.5-caused ATP decrease is rather
scarce. In this study, the influence of PM2.5 on energy metabolism in
SD rat lungs was investigated in order to explore the underlying
molecular mechanism of PM2.5-induced lung injury.
The most of ATP production under aerobic cellular respiration is
directly by oxidative phosphorylation in normal condition. In this
pathway, the substrates such as NADH and FADH2, necessary for
ATP synthesis through oxidative phosphorylation, are provided by
the TCA cycle. Therefore, the TCA cycle is considered as the up-
stream of oxidative phosphorylation and through this process total
36 ATP molecules are produced (Rueda et al., 2016). Nevertheless,
in stress condition the cytoplasmic glycolysis is triggered, thus
inducing the phosphorylation of the substrate level. In result 2 ATP
molecules are produced through transfer of phosphate groups from
1,3-biosphosphoglycerate and phosphoenolpyruvate to ADP
(Samanta and Semenza, 2017). It has been recognized that the
sustained occurrence of glycolysis can lead to the pathology and
ultrastructure alterations of lung tissues (Peronnet and Aguilaniu,
2014; Riede et al., 1981). Due to the importance of these meta-
bolic pathway in ATP production, we focused two major energy
pathways, the TCA cycle and glycolysis, to explain PM2.5 exposure
caused ATP depletion.
The PM2.5 samples were collected from Taiyuan city, a center of
coal-based electricity production and many chemical industries in
China, which currently is facing a serious articulate air pollution (Li
et al., 2014). Consequently, we aimed to evaluate the influence of
PM2.5 exposure from Taiyuan in summer and winter on ATP gen-
eration in lung tissues, and to analyse the possible mechanism of
energy metabolic rewiring. The data obtained will be helpful in
highlighting indicative biomarkers of PM2.5 exposure for energy
metabolism, and to help better understand the toxicological
mechanism of lung disease caused by particulate air pollution.
2. Materials and methods
2.1. PM2.5 preparation and characterization
As described in our previous study (Li et al., 2014), seasonal
PM2.5 samples were collected on quartz fiber filters (QFFs) for 22 h/
day using a PM2.5 high volume air sampler (Thermo Anderson, USA)
during 2012/2013 in Taiyuan. Then, these filters were cut and
submerged in Milli-Q water followed by sonication in order to
obtain PM2.5 powder samples. The obtained suspensions were
evaporated to dryness at 0  C under vacuum. The resulting powder
was weighed and stored in the dryer until used. 20 mg of PM2.5
were weighed, blended in 1 mL Milli-Q water followed by soni-
cation to obtain a 20 mg mL1 suspension. Before usage, the
suspension was treated with sonication. Particles were also UV-
irradiated for 2 h to prevent from possible contamination (Peeters
et al., 2014).
2.2. Animal experiment
Male Sprague-Dawley (SD) rats (200 ± 20 g) were commercially
purchased from Experimental Animal Center of the Chinese Mili-
tary Medical Science Academy in Beijing. SD rats were raised in
animal houses under a 12 h light/dark cycle at an invariable room
temperature. Then, they were randomly divided into seven groups
with six rats in each group after 7 days of habituation. The groups
included control group, three summer groups (0.2, 0.6, 1.5 mg kg1
body weight (b.w.)) and three winter groups (0.3, 1.5, 2.7 mg kg1 b.
w.). The control group was instilled with 0.5 mL physiological saline
solution, and PM2.5 groups with a 0.5 mL PM2.5 suspension to a final
exposure concentrations reached above using a nonsurgical intra-
tracheal instillation method (Bai et al., 2010). The exposure process
was performed once every third day for two continuous months. All
the animals were kept under standard environmental and nutri-
tional conditions according to the National Act on the Use of
Experimental Animals (China) requirements.
Summer and winter PM2.5 exposure doses were selected based
on the following information. It has been reported that the respi-
ratory volume of an adult rat was 200 mL min1 (Sung et al., 2016),
therefore the total respiratory volume of three days should be
0.864 m3. For example, on the basis of China National Ambient
Quality Standard for PM2.5 (0.075 mg m3), the PM2.5 exposure dose
for each rat (200 ± 20 g) every 3 days is estimated to be 0.324 mg
kg1 b. w. The ranges of PM2.5 mass concentrations monitored in
our sampling of summer and winter were 45.86e281.98 mg m3
and 42.13e692 mg m3, respectively (Li et al., 2014; Geng et al.,
2017). Hence, the PM2.5 exposure dose for each rat were esti-
mated in the scope from 0.215 to 1.512 mg kg1 b. w. for summer
PM2.5 and from 0.324 to 2.989 mg kg1 b. w. for winter PM2.5. 0.2,
0.6, 1.5 mg kg1 b. w. summer PM2.5 and 0.3, 1.5, 2.7 mg kg1 b. w.
winter PM2.5 were selected as exposure doses. It is worth
mentioning that 1.5 mg kg1 b. w. summer PM2.5 and 1.5 as well as
2.7 mg kg1 b. w. winter PM2.5 doses corresponded to the haze
weather, indicating the haze dosage.
2.3. Lung tissue preparation
After the animal euthanasia (Burkholder et al., 2010), lung tis-
sues were rapidly excised from the body, washed in ice-cold saline
solution, frozen in liquid nitrogen and weighed. The frozen tissues
were pulverized in the liquid nitrogen and subsequently homoge-
nized with a homogenizer either in a homogenization buffer for
western blot and enzyme activity analysis or in Trizol Reagent
(Invitrogen) for mRNA isolation. A BCA Assay Kit (Beyotime, Hai-
men, China) was used to determine the protein concentration of
lung tissue supernatant, obtained by the centrifugation of ho-
mogenates. The supernatants were quickly frozen at 80  C until
the detection of enzyme activity or protein levels.
2.4. Malondialdehyde (MDA) content
A lipid peroxidation MDA assay kit (Beyotime Institute of
Biotechnology, Beijing, China) was used to measure the lipid per-
oxidation content of lung tissue. Briefly, thiobarbituric acid (TBA) in
the kit was added to the lung supernatants and gently oscillated,
then the mixture was determined spectrophotometrically at
535 nm with four independent experiments. The MDA contents
were calculated in nmol mg1 protein (nmol mg1 prot).
 X. Jin et al. / Chemosphere 224 (2019) 29e38
2.5. ATP production
ATP production in the supernatant was quantified using an ATP
quantification kit, purchased from Nanjing Jiancheng Biological
Product (Nanjing, China). Following the manufacturer's in-
structions, the optical density value was measured using a fluo-
rescence microplate reader (Thermo Fisher Scientific, Franklin, MA,
USA) at 636 nm. ATP contents were calculated by the optical den-
sity value and standard concentrations. The data were expressed as
nmol mg1 protein.
2.6. qRT-PCR assay
Quantitative real time PCR (qRT-PCR) was carried out by an
Applied Bio-systems platform, as reported in our previous methods
(Jin et al., 2014). Primers for reference and targeted genes are
provided in Supplementary Table S1.
2.7. Western blotting assay
Tissues extracted proteins were resolved by SDS-PAGE and
transferred onto nitrocellulose membranes as described previously
(Jin et al., 2014). The antibodies used were as follows: antibody for
Glut1 (Bioworld Technology, Minneapolis, MN); antibody for PKM1
(Beyotime Institute of Biotechnology, Haimen, China); antibody for
GAPDH (Sigma, St. Louis, MO, USA).
2.8. Assessment of enzyme activities
The enzyme activities of citrate synthase (CS), malate dehy-
drogenase (MDH), hexokinase (HK) and pyruvate kinase (PKM)
were investigated using commercial kits (Nanjing Jiancheng Bio-
logical Product, Nanjing, China) Briefly, the enzyme reaction
mixture was prepared following the manufacturer's recommen-
dations and incubated for 15 min at 37  C. The reaction was initi-
ated by the addition of 200 mL of enzyme mixture and 10 mL lung
supernatant in a white 96-well plate. The optical density value was
immediately measured using a fluorescence microplate reader of
Thermo Fisher Scientific. Activities of MDH and HK were calculated
according to the change in optical density recorded at 1 or 2 min
intervals for 2 or 4 min at 340 nm, respectively. The activity of PKM
was calculated according to the change in optical density recorded
at 15 min intervals for 30 min at 340 nm. The activity of CS was
calculated according to the change in optical density recorded at an
interval of 15 min for 30 min at 412 nm. The activities of these
enzyme were calculated as U mg1 protein and U g1 protein.
2.9. Analysis of pyruvate and lactate production
Both pyruvate and lactate content were determined spectro-
photometrically (505 nm and 530 nm) using commercial kits
following the protocol provided by the manufacturer (Nanjing
Jiancheng Biological Product, Nanjing, China). The optical density
value was determined with a microplate reader. The pyruvate and
lactate levels were calculated as mmol g1 protein and mmol g1
protein, respectively.
2.10. Detection of tissue glucose content
Lung glucose levels were quantified using a glucose quantifi-
cation kit (Nanjing Jiancheng Biological Product, Nanjing, China).
Briefly, lung supernatant (10 mL) were transferred to an eppendorf
tube (EP) and reagents (1 mL) were added according to the man-
ufacturer's description. Absorbance measurements were per-
formed at 505 nm. The relative glucose levels in lung tissues were
31
calculated, and compared to the control group.
2.11. Immunohistochemical analysis
Immunohistochemistry (IHC) was employed following the
operation of Obert et al. (2007). One part of lung tissue was
immediately fixed in 10% formalin overnight, dehydrated and then
embedded in paraffin wax. As for the fixation process, we used a
10 mL glass vial full of 10% formalin to make sure the inflated lung
tissues were completely immersed in the fixation solution. The
section was used for histopathological examination and incubated
with the Glut1 antibody. After washing, they were incubated with
horseradish peroxidase-conjugated secondary antibody from
ZSGB-BIO, followed by staining with streptavidin-horseradish
peroxidase complex, diaminobenzidine (DAB), and hematoxylin.
2.12. Data analysis
Data analysis was performed using the SPSS 17.0 software pro-
gram. Values were shown as mean ± standard error (SE). Statistical
significance were analyzed by one-way analysis of variance
(ANOVA) according to the Tukey's post hoc test (*p < 0.05,
**p < 0.01).
3. Results
3.1. The effect of seasonal PM2.5 on ATP production in lung of SD rats
In order to investigate the impact of ambient PM2.5 on the ATP
generation in lung tissues, SD rats were sub-chronically exposed to
actual environmental dosage summer and winter PM2.5 (low, me-
dium and high) via the intratracheal instillation (Fig. 1A). Besides,
there was no significant variance on ATP generation and MDA
content of SD lungs between blank quartz fiber suspension and
physiological saline solution treatment at the same volume in our
preliminary experiments (data not shown). The organ coefficients
for lung in the summer groups (0.6 mg kg1 b. w. PM2.5) and winter
PM2.5 groups (0.3 mg kg1 b. w.) were obviously higher than that in
control group (Supplementary Table S2), suggesting the lung
swelling caused by non-haze PM2.5 and the lung damage occurred
by haze PM2.5. Concomitantly, contents of MDA, an indicator of lipid
peroxidation and tissue injury (Janero, 1990), were more conspic-
uously increased in haze PM2.5-exposed groups (Fig. 1B). Mean-
while, we observed suppression of ATP generation in lung tissues of
summer high dose PM2.5 and winter haze dose PM2.5, and found
that the injury of winter PM2.5 was more serious than summer
PM2.5 at the same dose (1.5 mg kg1 b. w.) (Fig. 1C). Therefore, haze
dosage PM2.5 exposure leads to the occurrence of lung tissue
damage and reduction of ATP content.
3.2. The activity of TCA cycle in lung tissues was weakened by PM2.5
exposure
Considering that TCA cycle is the major pathway of energy
metabolism in normal lung and the primary source of ATP (Date
et al., 1993), effects of ambient PM2.5 on TCA cycle in lung tissues
of SD rats were determined. The activities of CS and MDH, the
critical rate-limiting enzymes in TCA cycle, were performed using
commercially available kits. As presented in Fig. 2A, the CS activities
in summer high dose PM2.5 group and control group were 10.96 U
mg1 prot and 15.22 U mg1 prot, respectively. However, it was
slightly elevated upon low dose summer PM2.5 exposure, indicating
the stimulated action of non-haze dose PM2.5 on TCA cycle to resist
the disturbance caused by external stimulation (Clarke, 1993).
Furthermore, the reduced data found in the winter PM2.5 group
32 X. Jin et al. / Chemosphere 224 (2019) 29e38
Fig. 1. (A) Experimental protocol for PM2.5-exposed animals. (B) MDA content and (C) ATP generation in lung tissues. Values are expressed as the mean ± SE of 6 animals in each
group. *p < 0.05, **p < 0.01 vs control.
were more noticeable. With respect to the MDH activity, the data in
1.5 and 2.7 mg kg1 b. w. winter PM2.5 groups were 3.46 ± 0.29 U
mg1 prot and 2.69 ± 0.12 U mg1 prot, respectively. The data were
prominently lower for both groups (p < 0.01) than the control
group. Likewise, the promotion of MDH activity in low dose sum-
mer PM2.5 group (p < 0.05) suggested the resistance of low dosage
PM2.5-caused the lung stress response (Fig. 2B).
In the course of ATP biosynthesis, mitochondria can produce lots
of ATP for cells to live (Bagkos et al., 2014). Hence, we further
investigated the expressions of mitochondrial respiratory chain
genes, including NDUFS2, SDHD, UQCRI1 and COX4I1. As indicated
in Fig. 3, mRNA levels of UQCRI1 and NDUFS2 were notably
declined by summer low dose PM2.5 or winter haze dose PM2.5
exposure. No prominent difference between control groups and
PM2.5 groups was found on SDHD and COX4I1 expressions
(Fig. 3CeD). In summary, haze dose of PM2.5 exposure reduces the
ATP generation in lung via the attenuation of key enzyme in TCA
cycle and the down-regulated expression of electron transport
chain complex I and III.
3.3. The level of glycolysis in lung tissues was strengthened in
response to PM2.5
To comprehensively explore the influence of PM2.5 exposure on
energy metabolism, the glycolysis, another main energy meta-
bolism (Riede et al., 1981), were further analyzed in lung tissues.
The contents of metabolite in glycolysis, including pyruvate and
lactic acid, were measured firstly. As shown in Fig. 4A and B, levels
of pyruvate and lactic acid in rat lungs markedly elevated in PM2.5
groups as summer and winter PM2.5 doses increased. The consis-
tent elevation was observed in enzyme activities of hexokinase
(HK) and pyruvate kinase (PKM), which catalyses the glucose
glycolysis reaction (Fig. 4CeD). Regarding the PKM activity, winter
high dose PM2.5 group was 413.26 ± 29.23 U g1 prot, which was
considerably higher than that control group. Correspondingly, the
PKM mRNA level was increased in the PM2.5 groups (Fig. 4E). With
regard to the protein expression of PKM, the result was in line with
the PKM mRNA level (Fig. 4F). Lactate dehydrogenase (LDH) is an
important regulatory enzyme that produces lactic acid in glycolysis.
The expressions of LDH, including subunits LDHA and LDHB, were
considerably increased as seasonal PM2.5 doses raised (Fig. 4GeH).
It therefore concludes that sub-chronic exposure to haze dosage
PM2.5 reduced the ATP generation in lung via the metabolic con-
version in lung from TCA cycle to glycolysis.
3.4. The influence of glucose uptake and its transporter exposed to
PM2.5
As the major energy substrate, alteration of glucose contents in
lung tissues has a primary role in the glycolysis regulation (Bonora
et al., 2012). Fig. 5A illustrated an inconspicuous alteration of
glucose uptake in PM2.5-exposed lung tissues of SD rats. However,
mRNA level of glucose transporter 1 (Glut1), which can regulate the
glucose content of lung tissues, was increased upon haze dosage
PM2.5 exposure (Fig. 5B). In particular, the relative Glut1 expres-
sions in summer and winter 1.5 mg kg1 b. w. PM2.5 groups were
about 1.91 ± 0.07 and 2.18 ± 0.08, compared to control group. Ul-
teriorly, the protein levels of Glut1 in 1.5 mg kg1 b. w. seasonal
PM2.5 groups were compared. As shown in Fig. 5C, the up-
regulation of Glut1 in winter PM2.5 groups was more noticeable.
In addition, IHC was carried out to confirm the alteration of Glut1
expression in lung tissues, and the result was in line with the trend
from qRT-PCR assay (Fig. 5D). These results imply that haze dose
PM2.5 exposure elevates Glut1 expressions in lung tissues of SD rats,
which administers the stimulated occurrence of glycolysis.
 X. Jin et al. / Chemosphere 224 (2019) 29e38 33

Fig. 2. Enzyme activities of (A) CS and (B) MDH in TCA cycle in lung tissues upon PM2.5 exposure. Error bars indicated the means ± SE of triplicate determinations. *p < 0.05,
**p < 0.01, compared to control group.

4. Discussion ATP content than the corresponding dose in summer samples,

In the current study, an ATP depletion was observed in PM2.5-
exposed rat lungs, suggesting that the energy homeostasis and
functional activity of the organ were disordered by the sub-chronic
exposure of haze dosage PM2.5. Similarly, Thomson et al. reported
an interesting data, showing the decrease of ATP production upon
PM2.5 exposure in human A549 lung epithelial cells (Thomson et al.,
2015). Furthermore, PM2.5-caused ATP reduction and ATP-binding
cassette (ABC) transporter activity in brain mitochondria (Ku
et al., 2016) and cardiac tissues (Ribeiro Jde et al., 2016) were also
determined. More specifically, significant ATP reduction was
observed in winter high dosage PM2.5 (2.7 mg kg1 b. w.) group,
while winter medium dose PM2.5 (1.5 mg kg1 b. w.) exhibited less
although the differences were not statistically significant.
The seasonal variation may correlate with both particle mass
concentrations and chemical compositions, which were strongly
influenced by the source of PM2.5 including biomass and coal
burning, coal burning emissions, and vehicle exhaust (Li et al.,
2014; Cui et al., 2016; Liu et al., 2015). On the one hand, haze
weather in Taiyuan winter is much higher and more serious than in
summer, and the doses in our study were selected according to the
range of actual atmospheric seasonal PM2.5 concentration. On the
other hand, chemical compositions and enrichment factors in PM2.5
varied greatly in different seasons and can affect the PM2.5 toxicity.
The concentration of organic component (OC) and elemental car-
bon (EC) in summer particulate matter were lower than that in
34 X. Jin et al. / Chemosphere 224 (2019) 29e38
Fig. 3. The expressions of mitochondrial respiratory chain complexes, including (A) NDUFS2, (B) SDHD, (C) UQCRI1 and (D) COX4I1 in lung tissues. Data were mean ± SE of three
identical experiments. *p < 0.05, **p < 0.01.
winter (Li et al., 2014; Sung et al., 2016). It is increasingly recognized
that organic compound enriched in winter PM2.5 is responsible for
the seasonal difference. For example, Longhin et al. indicated that
winter PM-caused damage responses in BEAS-2B cells were
apparently related to the high content of organic compounds, while
effects of summer PM may be associated with biological and inor-
ganic components (Longhin et al., 2016). Compared to summer PM,
ROS and DNA damage in A549 cells and THP-1 cells were caused
only by winter PM fractions, and the winter PM damaging effect on
DNA correlated with the presence of organic compounds (Longhin
et al., 2013). In literature, some researches also reported that PM2.5-
heavy metals caused lung injury. Stephen et al. indicated that metal
composition of ambient PM2.5 influenced severity of allergic air-
ways disease in mice (Stephen et al., 2003). Additionally, metal-rich
PM2.5 can also lead to airway inflammation in healthy subjects
(Frank et al., 2004). As a matter of fact, endotoxin is also a non-
negligible component in airborne particulate matters, and we
tried to evaluate the actual deleterious effects in lung tissues after
the exposure of PM2.5 containing complex compositions. Therefore,
the metabolic reprogramming in exposed rat lung tissues observed
in this work could be contributed by the combined effects of many
compositions in PM2.5.
The level of cellular ATP may be regulated by the coordinated
activity of energy substrate utilization and ATP synthesis, which
might include the main energy substrate content and transporter,
TCA cycle and glycolysis pathway, mitochondrial respiratory chain
and so on. One of the primary metabolic alteration discovered in
lung tissue was an increased metabolic rearrangement from TCA
cycle to glycolysis, implying metabolic abnormality in lung tissues.
Nevertheless, there are sparse studies concerning the disruption of
metabolic pathway caused by PM2.5 exposure. A proteomic study
from PM2.5-exposed A549 cells showed that metabolic disturbance
was one of major factors contributing to PM2.5-mediated toxicity in
human lung cells (Huang et al., 2014). Recently, Zhang et al. also
pointed out that TCA cycle was a main metabolic pathway affected
by PM2.5 exposure (Zhang et al., 2017). Recent data from Liu et al.
suggested that PM2.5 could directly dysregulate the glucose meta-
bolism (Liu et al., 2017). Besides, an impairment of whole-body
glucose tolerance and Glut4 reduction in response to PM2.5 in
skeletal muscle were also observed (Liu et al., 2014).
Additionally, the expressions of electron transport chain genes, I
and III, were significantly inhibited by summer PM2.5 (1.5 mg kg1
b. w.) or winter PM2.5 (1.5 and 2.7 mg kg1 b. w.) exposure. This
effect was in accordance with our previous study, showing that
expressions of mitochondrial respiratory chain genes in 16HBE
cells, including NDUFS2 and UQCRI1, were also attenuated by PM2.5
exposure (Jin et al., 2016). The alterations of mitochondrial mem-
brane potential and morphology due to PM2.5 exposure using both
JC-1 and MitoTracker Red staining were also observed in our pre-
vious study (Jin et al., 2016). Mitochondrial redox homeostasis in
healthy and chronic obstructive pulmonary diseased human
bronchial epithelial cells can be critically impaired by PM2.5, which
may produces a persistent mitochondrial dysfunction and a
lowering cell energy supply (Leclercq et al., 2018). Moreover,
Soberanes et al. indicated that the site III of mitochondrial electron
transport chain was primarily responsible for the PM2.5-induced
oxidant production (Soberanes et al., 2009). Under conditions of
reduced O2 availability, electron transport becomes less efficient,
leading to increased generation of superoxide anions, and hypoxia-
inducible factors switch cells from oxidative to glycolytic meta-
bolism (Samanta and Semenza, 2017). Therefore, O2 availability
decreased by PM2.5 exposure may be one causation for the meta-
bolic transformation in lung tissues. Byun et al. demonstrated that
blood mtDNA methylation was negatively associated with PM2.5
exposure, suggesting the mitochondrial dysfunction and adverse
health outcome caused by ambient fine particles (Byun et al., 2016).
 X. Jin et al. / Chemosphere 224 (2019) 29e38 35

Fig. 4. Contents of (A) pyruvate and (B) lactic acid in rat lung upon PM2.5 exposure. Enzyme activities of (C) HK and (D) PKM. (E) mRNA and (F) protein levels of PKM1. Expressions of
LDH, including subtypes (G) LDHA and (H) LDHB. Data were mean ± SE of triplicate determinations. *p < 0.05, **p < 0.01 vs control.

To our knowledge, this is the novel report to examine the effect contributing to the ATP reduction (Fig. 6). The metabolic alteration
of PM2.5 exposure on metabolic reprogramming of lung tissues. The is orchestrated by PM2.5 exposure and may conduct to the devel-
data implicated that sub-chronic exposure to ambient PM2.5 led to opment of lung diseases. Accordingly, this study shows a possible
the attenuation of TCA cycle and elevation of glycolytic levels, mechanism of lung diseases induced by haze PM2.5, providing
36 X. Jin et al. / Chemosphere 224 (2019) 29e38

Fig. 5. Impact of (A) glucose uptake and (B) mRNA levels of Glut1 upon PM2.5 exposure. (C) The comparison of summer and winter PM2.5 (1.5 mg kg1 b. w.) on Glut1 expressions.
Values are expressed as the mean ± SE (n ¼ 3). *p < 0.05, **p < 0.01 vs control. (D) Glut1 content in lung tissues by the IHC analysis (20  magnification).

Fig. 6. The proposed model of ambient fine particles exposure leading to the abnormal energy metabolism and ATP decrease in lung tissues.
 X. Jin et al. / Chemosphere 224 (2019) 29e38
indicative and useful biomarkers of inhalable PM2.5 exposure, and
assisting the development of preventive treatments for urban dust-
haze.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (grant number 21707085 and 31770382), the
China Postdoctoral Science Foundation (grant number
2017M610120), the Shanxi Province Science Foundation for Youths
(No. 201701D221244), the Fund for Shanxi “1331 Project” Collab-
orative Innovation Center (No. 1331 CIC) and the Shanxi Scholarship
Council of China (grant number 2015-2).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2019.02.116.
Financial and competing interest disclosure
The authors declare they have no actual or potential competing
financial interests.
References
Bagkos, G., Koufopoulos, K., Piperi, C., 2014. ATP synthesis revisited: new avenues
for the management of mitochondrial diseases. Curr. Pharmaceut. Des. 20,
4570e4579.
Bai, R., Zhang, L.L., Liu, Y., Meng, L., Wang, L.M., Wu, Y., Li, W., Ge, C.C., Le Guyader, L.,
Chen, C.Y., 2010. Pulmonary responses to printer toner particles in mice after
intratracheal instillation. Toxicol. Lett. 199, 288e300.
Bonora, M., Patergnani, S., Rimessi, A., De Marchi, E., Suski, J.M., Bononi, A.,
Giorgi, C., Marchi, S., Missiroli, S., Poletti, F., Wieckowski, M.R., Pinton, P., 2012.
ATP synthesis and storage. Purinergic Signal. 8, 343e357.
Brunekreef, B., Holgate, S.T., 2002. Air pollution and health. Lancet 360, 1233e1242.
Burkholder, T.H., Niel, L., Weed, J.L., Brinster, L.R., Bacher, J.D., Foltz, C.J., 2010.
Comparison of carbon dioxide and argon euthanasia: effects on behavior, heart
rate,and respiratory lesions in rats. J. Am. Assoc. Lab. Anim. Sci. 49, 448e453.
Byun, H.M., Colicino, E., Trevisi, L., Fan, T., Christiani, D.C., Baccarelli, A.A., 2016.
Effects of air pollution and blood mitochondrial DNA methylation on markers of
heart rate variability. J. Am. Heart Assoc. 5, e003218.
Clarke, A., 1993. Seasonal acclimatization and latitudinal compensation in meta-
bolism: Do they exist? Funct. Ecol. 2, 139e149.
Cui, J.L., Zhang, Z.H., Xia, N., Ping, F.F., Geng, H., 2016. Pollution characteristics of
heavy metals in PM2.5 during four seasons in Taiyuan City. Acta Sci. Circum-
stantiae 5, 1566e1572.
Date, H., Matsumura, A., Manchester, J.K., Obo, H., Lima, O., Cooper, J.M.,
Sundaresan, S., Lowry, O.H., Cooper, J.D., 1993. Evaluation of lung metabolism
during successful twenty-four-hour canine lung preservation. J. Thorac. Car-
diovasc. Surg. 105, 480e491.
Frank, S., Paul, A.B., Andreas, H., Johannes, K., Mike, P., Roel, S., Birgit, L., Jens, H.,
Joachim, H., Norbert, K., 2004. Metal-rich ambient particles (particulate Mat-
ter2.5) cause airway inflammation in healthy subjects. Am. J. Respir. Crit. Care
Med. 170, 898e903.
Gehring, U., Gruzieva, O., Agius, R.M., Beelen, R., Custovic, A., Cyrys, J., Eeftens, M.,
Flexeder, C., Fuertes, E., Heinrich, J., Hoffmann, B., de Jongste, J.C., Kerkhof, M.,
Klumper, C., Korek, M., Molter, A., Schultz, E.S., Simpson, A., Sugiri, D.,
Svartengren, M., von Berg, A., Wijga, A.H., Pershagen, G., Brunekreef, B., 2013.
Air pollution exposure and lung function in children: the ESCAPE project. En-
viron. Health Perspect. 121, 1357e1364.
Geng, H., Jin, C.S., Zhang, D.P., Wang, S.R., Xu, X.T., Zhang, Y., Ro, C.U., 2017. Char-
acterization of size-resolved urban haze particles collected in summer and
winter at Taiyuan City, China using quantitative electron probe X-ray micro-
analysis. Atmos. Res. 190, 29e42.
Gotschi, T., Heinrich, J., Sunyer, J., Kunzli, N., 2008. Long-term effects of ambient air
pollution on lung function: a review. Epidemiology 19, 690e701.
Grattagliano, I., Bonfrate, L., Diogo, C.V., Wang, H.H., Wang, D.Q., Portincasa, P., 2009.
Biochemical mechanisms in drug-induced liver injury: certainties and doubts.
World J. Gastroenterol. 15, 4865e4876.
Huang, Q.Y., Zhang, J., Peng, S.Y., Tian, M.P., Chen, J.S., Shen, H.Q., 2014. Effects of
water soluble PM2.5 extracts exposure on human lung epithelial cells (A549): a
proteomic study. J. Appl. Toxicol. 34, 675e687.
Janero, D.R., 1990. Malondiadehyde and thiobarbituric acid-reactivity as diagnostic
indices of lipid peroxidation and peroxidative tissue injury. Free Radic. Biol.
Med. 9, 515e540.
Jin, X.T., Chen, M.L., Song, L., Li, H.Q., Li, Z.Y., 2014. The evaluation of p, p0 -DDT
37
exposure on cell adhesion of hepatocellular carcinoma. Toxicology 322, 99e108.
Jin, X.T., Su, R.J., Li, R.J., Song, L., Chen, M.L., Cheng, L., Li, Z.Y., 2016. Amelioration of
particulate matter-induced oxidative damage by vitamin c and quercetin in
human bronchial epithelial cells. Chemosphere 144, 459e466.
Ku, T.T., Ji, X.T., Zhang, Y.Y., Li, G.K., Sang, N., 2016. PM2.5, SO2 and NO2 co-exposure
impairs neurobehavior and induces mitochondrial injuries in the mouse brain.
Chemosphere 163, 27e34.
Leclercq, B., Kluza, J., Antherieu, S., Sotty, J., Alleman, L.Y., Perdrix, E., Loyens, A.,
Coddeville, P., Lo Guidice, J.M., Marchetti, P., Garçon, G., 2018. Air pollution-
derived PM2.5 impairs mitochondrial function in healthy and chronic obstruc-
tive pulmonary diseased human bronchial epithelial cells. Environ. Pollut. 243,
1434e1449.
Liu, C.Q., Xu, X.H., Bai, Y.T., Wang, T.Y., Rao, X., Wang, A.X., Sun, L.X., Ying, Z.K.,
Gushchina, L., Maiseyeu, A., Morishita, M., Sun, Q.H., Harkema, J.R.,
Rajagopalan, S., 2014. Air pollution-mediated susceptibility to inflammation
and insulin resistance: influence of CCR2 pathways in mice. Environ. Health
Perspect. 122, 17e26.
Li, R.J., Kou, X.J., Geng, H., Dong, C., Cai, Z.W., 2014. Pollution characteristics of
ambient PM2.5-bound PAHs and NPAHs in a typical winter time period in
Taiyuan. Chin. Chem. Lett. 25, 663e666.
Liu, C.Q., Xu, X.H., Bai, Y.T., Zhong, J.X., Wang, A.X., Sun, L.X., Kong, L.Y., Ying, Z.K.,
Sun, Q.H., Rajagopalan, S., 2017. Particulate Air pollution mediated effects on
insulin resistance in mice are independent of CCR2. Part. Fibre Toxicol. 14, 6.
Liu, S., Peng, L., Wen, Y.P., Bai, H.L., Liu, F.X., Shi, M.X., Liu, F.X., Li, L.J., 2015. Pollution
characteristics of organic and elemental carbon in PM2.5 in Taiyuan. Environ.
Sci. 2, 1566e1572.
Longhin, E., Capasso, L., Battaglia, C., Proverbio, M.C., Cosentino, C., Cifola, I.,
Mangano, E., Camatini, M., Gualtieri, M., 2016. Integrative transcriptomic and
protein analysis of human bronchial BEAS-2B exposed to seasonal urban par-
ticulate matter. Environ. Pollut. 209, 87e98.
Longhin, E., Pezzolato, E., Mantecca, P., Holme, J.A., Franzetti, A., Camatini, M.,
Gualtieri, M., 2013. Season linked responses to fine and quasi-ultrafine Milan
PM in cultured cells. Toxicol. Vitro 27, 551e559.
Meng, Z., Zhang, Q., 2006. Oxidative damage of dust storm fine particles instillation
on lungs, hearts and livers of rats. Environ. Toxicol. Pharmacol. 22, 277e282.
Obert, L.A., Sobocinski, G.P., Bobrowski, W.F., Metz, A.L., Rolsma, M.D.,
Altrogge, D.M., Dumstan, R.W., 2007. An immunohistochemical approach to
differentiate hepatic lipidosis from hepatic phospholipidosis in rats. Toxicol.
Pathol. 35, 728e734.
Peeters, P.M., Eurlings, I.M., Perkins, T.N., Wouters, E.F., Schins, R.P., Borm, P.J.,
Drommer, W., Reynaert, N.L., Albrecht, C., 2014. Silica-induced NLRP3 inflam-
masome activation in vitro and in rat lungs. Cancer 3, 4.
Peronnet, F., Aguilaniu, B., 2014. Physiological significance and interpretation of
plasma lactate concentration and pH in clinical exercise testing. Rev. Mal.
Respir. 31, 525e551.
Pierdominici, M., Maselli, A., Cecchetti, S., Tinari, A., Mastrofrancesco, A., Alfe, M.,
Gargiulo, V., Beatrice, C., Di Blasio, G., Carpinelli, G., Ortona, E., Giovannetti, A.,
Fiorito, S., 2014. Diesel exhaust particle exposure in vitro impacts T lymphocyte
phenotype and function. Part. Fibre Toxicol. 11, 74.
Ribeiro Jde, P., Kalb, A.C., Campos, P.P., Cruz, A.R., Martinez, P.E., Gioda, A.,
Souza, M.M., Gioda, C.R., 2016. Toxicological effects of particulate matter (PM2.5)
on rats: bioaccumulation, antioxidant alterations, lipid damage, and ABC
transporter activity. Chemosphere 163, 569e577.
Riede, U., Sandritter, W., Mittermayer, C., 1981. Circulatory shock: a review. Pa-
thology 13, 299e311.
Rueda, E.M., Johnson Jr., J.E., Giddabasappa, A., Swaroop, A., Brooks, M.J., Sigel, I.,
Chaney, S.Y., Fox, D.A., 2016. The cellular and compartmental profile of mouse
retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P
transferring kinases. Mol. Vis. 22, 847e885.
Samanta, D., Semenza, G.L., 2017. Maintenance of redox homeostasis by hypoxia-
inducible factors. Redox. Biol. 13, 331e335.
Senthilkumar, S., Manju, A., Muthuselvam, P., Shalini, D., Indhumathi, V.,
Kalaiselvi, K., Palanivel, M., Chandrasekar, P.P., Rajaguru, P., 2014. Character-
ization and genotoxicity evaluation of particulate matter collected from in-
dustrial atmosphere in Tamil Nadu state, India. J. Hazard Mater. 274, 392e398.
Sharma, G., Goodwin, J., 2006. Effect of aging on respiratory system physiology and
immunology. Clin. Interv. Aging 1, 253e260.
Soberanes, S., Urich, D., Baker, C.M., Burgess, Z., Chiarella, S.E., Bell, E.L., Ghio, A.J., De
Vizcaya-Ruiz, A., Liu, J., Ridge, K.M., Kamp, D.W., Chandel, N.S., Schumacker, P.T.,
Mutlu, G.M., Budinger, G.R., 2009. Mitochondrial complex III-generated oxi-
dants activate ASK1 and JNK to induce alveolar epithelial cell death following
exposure to particulate matter air pollution. J. Biol. Chem. 284, 2176e2186.
Stephen, H.G., Najwa, H.C., Lisa, B.C., Joachim, H., Gilmour, M.I., 2003. Metal
composition of ambient PM2.5 influences severity of allergic airways disease in
mice. Environ. Health Perspect. 111, 1471e1477.
Sung, K., Hyelim, P., Hyewon, P., Boyoung, J., Eunkyung, K., 2016. The acute respi-
ratory exposure by intratracheal instillation of SpragueeDawley rats with diesel
particulate matter induces retinal thickening. Cutan. Ocul. Toxicol. 35, 275e280.
Thomson, E.M., Breznan, D., Karthikeyan, S., MacKinnon-Roy, C., Charland, J.P.,
Dabek-Zlotorzynska, E., Celo, V., Kumarathasan, P., Brook, J.R., Vincent, R., 2015.
Cytotoxic and inflammatory potential of size-fractionated particulate matter
collected repeatedly within a small urban area. Part. Fibre Toxicol. 12, 24.
Wu, S.W., Deng, F.R., Hao, Y., Shima, M., Wang, X., Zheng, C.J., Wei, H.Y., Lv, H.B.,
Lu, X.L., Huang, J., Qin, Y., Guo, X.B., 2013. Chemical constituents of fine par-
ticulate air pollution and pulmonary function in healthy adults: the Healthy
38 X. Jin et al. / Chemosphere 224 (2019) 29e38

Volunteer Natural Relocation study. J. Hazard Mater. 260, 183e191.
Xing, Y.F., Xu, Y.H., Shi, M.H., Lian, Y.X., 2016. The impact of PM2.5 on the human
respiratory system. J. Thorac. Dis. 8, E69eE74.
Xu, T., Zhao, J., Hu, P., Dong, Z.J., Li, J.Y., Zhang, H.C., Yin, D.Q., Zhao, Q.S., 2014.
Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos
at gastrulation stage. Toxicol. Appl. Pharmacol. 277, 183e191.
Zhang, Y.N., Hu, H.J., Shi, Y.F., Yang, X.Z., Cao, L.G., Wu, J., Asweto, C.O., Feng, L.,
Duan, J.C., Sun, Z.W., 2017. 1H NMR-based metabolomics study on repeat dose
toxicity of fine particulate matter in rats after intratracheal instillation. Sci. Total
Environ. 589, 212e221.