Effect of Ambient PM2.5 on Lung Mitochondrial Damage and Fusion

Article pubs.acs.org/crt

Effect of Ambient PM2.5 on Lung Mitochondrial Damage and Fusion/ Fission Gene Expression in Rats Ruijin Li,† Xiaojing Kou,† Hong Geng,† Jingfang Xie,† Zhenhua Yang,† Yuexia Zhang,† Zongwei Cai,*,‡ and Chuan Dong*,† †

Institute of Environmental Science, College of Environmental & Resource Sciences, Shanxi University, Taiyuan 030006, China State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China

‡

ABSTRACT: Exposure to ambient fine particulate matter (PM2.5) increases the risk of respiratory disease. Although previous mitochondrial research has provided new information about PM toxicity in the lung, the exact mechanism of PM2.5mediated structural and functional damage of lung mitochondria remains unclear. In this study, changes in lung mitochondrial morphology, expression of mitochondrial fission/fusion markers, lipid peroxidation, and transport ATPase activity in SD rats exposed to ambient PM2.5 at different dosages were investigated. Also, the release of reactive oxygen species (ROS) via the respiratory burst in rat alveolar macrophages (AMs) exposed to PM2.5 was examined by luminoldependent chemiluminescence (CL). The results showed that (1) PM2.5 deposited in the lung and induced pathological damage, particularly causing abnormal alterations of mitochondrial structure, including mitochondrial swelling and cristae disorder or even fragmentation in the presence of higher doses of PM2.5; (2) PM2.5 significantly affected the expression of specific mitochondrial fission/fusion markers (OPA1, Mfn1, Mfn2, Fis1, and Drp1) in rat lung; (3) PM2.5 inhibited Mn superoxide dismutase (MnSOD), Na+K+-ATPase, and Ca2+-ATPase activities and elevated malondialdehyde (MDA) content in rat lung mitochondria; and (4) PM2.5 induced rat AMs to produce ROS, which was inhibited by about 84.1% by diphenyleneiodonium chloride (DPI), an important ROS generation inhibitor. It is suggested that the pathological injury observed in rat lung exposed to PM2.5 is associated with mitochondrial fusion−fission dysfunction, ROS generation, mitochondrial lipid peroxidation, and cellular homeostasis imbalance. Damage to lung mitochondria may be one of the important mechanisms by which PM2.5 induces lung injury, contributing to respiratory diseases.

1. INTRODUCTION Mammalian mitochondria are one of the most important organelles and play key roles in energy production, cell metabolism, programmed cell death, calcium homeostasis, and other biochemical functions.1 Mitochondrial dysfunction may contribute to the pathogenesis of lung diseases such as asthma, cystic fibrosis, and lung cancer.2,3 Mitochondria are morphologically dynamic organelles that continuously divide and fuse to form small individual units or interconnected networks within the cell,1 and mitochondrial fusion and fission are essential for maintaining normal cell functions.4 It has been reported that mitochondrial fusion is mediated by optic atrophy protein 1 (OPA1), mitofusin (Mfn) 1, and Mfn2, whereas fission is regulated by several proteins such as dynamin-related protein 1 (Drp1) and fission-mediator protein 1 (Fis1).1−3 Disorder in the balance of mitochondrial fission and fusion may result in abnormal changes in mitochondrial structure and function, which are associated with respiratory diseases.2,3 Ambient particulate matter with an aerodynamic diameter of less than 2.5 μm (PM2.5) mostly originates from coal combustion, vehicle exhaust, construction, and agricultural pollution.5 Over recent decades, many epidemiological studies © 2015 American Chemical Society

have highlighted the role of ambient PM2.5 as a major environmental pollutant that greatly contributes to respiratory diseases, e.g., asthma and lung cancer.6−8 Moreover, increasing evidence has indicated that the production of reactive oxygen species (ROS), oxidative stress, inflammation, and the formation of DNA adducts are involved in PM2.5-mediated lung injury.9−11 It is well-known that the mitochondrion is a sensitive target of both oxidative stress and environmental toxicants like PM2.5.12,13 PM2.5 may induce mitochondrial damage in exposed individuals,14 and PM-induced effects on the respiratory tract may be mediated partially by mitochondrial dysfunction.3,15 Because disruption in the fission−fusion process is an important regulatory mechanism of mitochondrial dysfunction and because only limited data are available on the molecular mechanisms of PM2.5 on the balance of fusion/fission in lung mitochondria, elucidating the expression pattern of fusion and fission genes after PM2.5 exposure will be useful for Special Issue: Chemical Toxicology in China Received: September 10, 2014 Published: January 5, 2015 408

DOI: 10.1021/tx5003723 Chem. Res. Toxicol. 2015, 28, 408−418

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Chemical Research in Toxicology

groups of five animals each: (1) control group, (2) 0.375 mg/kg bw PM2.5 group, (3) 1.5 mg/kg bw PM2.5 group, (4) 6.0 mg/kg bw PM2.5 group, and (5) 24.0 mg/kg bw PM2.5 group. The control group was instilled with physiological saline at the same volume as that used for the treatment groups, and the other special control group (vehicle group) was treated with the same volume of a suspension from extracts from a blank filter. The other four treatment groups were instilled with a PM2.5 suspension at different concentrations, and final the exposure concentration reached 0.375, 1.5, 6.0, and 24.0 mg/kg bw, respectively. The instillation was performed using a nonsurgical intratracheal instillation method adapted from ref 19. The volume of instillation of physiological saline or PM2.5 suspension was 0.5 mL. Instillation was performed five times every 2 days. When not being treated, the rats had free access to food and water. The care and use of the animals reported in this study was approved by the Institutional Animal Care and Use Committee of Shanxi University. 2.5. SEM and TEM Analyses. The rats were euthanized using sodium pentobarbital (80 mg/kg bw, i.p.) 24 h after the last treatment. Then, lungs were removed and washed in ice-cold phosphate buffer solution (PBS) to remove the superficial blood. A piece of the lung was cut and fixed in 4% paraformaldehyde in PBS, and then the samples were dried and dehydrated. The adhesion status of PM2.5 in the lung tissue samples was investigated by a field emission scanning electron microscope (FESEM LEO 1530 VP, Germany). At the same time, another piece of lung tissue was minced into appropriate small fragments (about 1 mm3), 4−6 of which were immediately fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.2−7.4) and postfixed in 1% of buffered osmium tetroxide. Then, the samples were dehydrated in ethanol, embedded in fresh Epon in capsules, and polymerized at 60 °C for 48 h. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined and photographed under a JEOL-100 CX instrument (JEOL, Tokyo) operating at an accelerating voltage of 80 kV. Under 20 000× magnification by TEM, PM2.5 deposition in the lung was observed and graded semiquantitatively as − (no particles), ∓ (very few particles), + (small amount of particles), ++ (moderate amount of particles), or +++ (relatively more particles), based on the relative amounts of PM deposition. Also, the degree of mitochondrial damage in the different groups was estimated according to the mitochondrial damage classification method described by Flameng et al.20 In brief, under the same magnification, 20 mitochondria from each of 5 randomly selected fields (100 mitochondria per rat) were analyzed. The mitochondrial damage types were assessed using a scale from 0 to 4, with 0 indicating a normal structure, 1, normal with slight swelling, 2, mitochondrial swelling, 3, serious swelling and cristae disorder, and 4, membrane breach, vacuolization, and fragmentation. On the basis of the above scale, the degree of mitochondrial damage in the different groups of rats was scored, and the total scores of 100 mitochondria were summed. Finally, this value was divided by 100, and the ratio accounted for the degree of mitochondrial damage. The higher the ratio, the more severe is the damage. 2.6. Real-Time Quantitative RT-PCR. After the last treatment, rats were sacrificed, and then some lung tissue was quickly frozen in liquid nitrogen and stored at −80 °C. When performing RT-PCR analysis, the frozen lung tissue samples were thawed, and total RNA was extracted using the Transzol reagent (Transgen, Beijing, China). Then, first-strand cDNA was synthesized using an AMV RT firststrand cDNA synthesis kit (Transgen, Beijing, China) according to the manufacturer’s protocols. cDNA was stored at −80 °C until use. The mRNA level of fusion and fission related genes was assessed by real-time PCR in a iCycler iQ real-time PCR detection system (BioRad, Hercules, CA, USA) with the SYBR Premix Ex Taq (perfect real time) kit (TaKaRa, Dalian, China). PCR amplification was performed in a 20 μL reaction solution containing 7 μL of ddH2O, 10 μL of SYBR Premix Ex Taq, 1 μL each of the forward and reverse primers, and 1 μL of cDNA using the iCycler iQ real-time PCR detection system. Thermal cycling was carried out as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 94 °C for 20 s, 55 °C (for OPA1, Drp1, Fis1, and actin) or 60 °C (for Mfn1 and Mfn2) for 20 s, and 72 °C for 20 s. Melting curve analysis was also performed using 81 cycles of 15 s increasing from 55 to 95 °C. The relative quantification

understanding the mechanisms of PM2.5-induced toxicological effects in the lung. This study investigates the lung injury effects of urban winter PM2.5 on rats in relation to alterations in the expression of mitochondrial fission and fusion genes, oxidative stress, and Na+K+-ATPase and Ca2+-ATPase activities. The experiments performed include (1) measuring the chemical components of PM, checking the deposition characteristic of PM2.5 in rat lungs using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and examining the ultrastructural changes in the exposed lungs and mitochondria; (2) analyzing the mRNA expression of specific mitochondrial fission/fusion markers (OPA1, Mfn1, Mfn2, Drp1, and Fis1) in rat lung using real-time RT-PCR and the protein levels in lung mitochondria using western blot; and (3) detecting the levels of Mn superoxide dismutase (MnSOD), Na+K+-ATPase, Ca2+-ATPase, and malondialdehyde (MDA) in rat lung mitochondria as well as ROS production in rat alveolar macrophages (AMs) exposed to PM2.5.

2. MATERIALS AND METHODS 2.1. PM2.5 Sample Collection. The sampling site was located on the roof of a five-story building (about 25 m above ground) on the Shanxi University campus (30 °15′ N, 112° 33′ E) in Taiyuan, China, during January, 2013. Taiyuan, the capital of Shanxi, China, is a center for energy production and chemical industries and is currently facing serious air pollution issues in the winter with extremely high concentrations of atmospheric PM2.5. PM2.5 mass concentrations were measured using a DustTrak II aerosol monitor (TSI Inc., USA), and daily PM2.5 samples were collected on quartz fiber filters (QFFs) for 24 h/day using a PM2.5 high-volume air sampler (Thermo Anderson, USA), with a pump flow rate of 1.13 m3/min. The filters used were prebaked at 450 °C for 6 h and equilibrated in desiccators. The QFFs after sampling were packed in clean aluminum foil and stored at −20 °C until use. 2.2. Chemical Analysis. The concentrations of polycyclic aromatic hydrocarbon (PAHs) on PM2.5 during sampling were measured by gas chromatograph−mass spectrometer (GC−MS), and the concentrations of nitrate (NO3−) and sulfate (SO42−) were analyzed by ion chromatography. 2.3. Preparation of PM2.5 Suspension and Selection of Exposure Concentrations. Five sheets of QFFs loaded with PM2.5 during the sampling time and one blank QFF were cut and submerged in Milli-Q water with sonication. The extraction was filtered through six layers of sterile gauze, and then the collecting solution was obtained and freeze-dried in vacuum. Prior to use, the dried samples were mixed, weighed, and then diluted in sterilized 0.9% physiological saline with swirling for 10 min. As has been reported, the respiratory volume of an adult rat is 200 mL/min, and the respiratory volume for 2 days reaches 0.576 m3. According to the China National Ambient Quality Standard (NAAQS, 2012) for PM2.5 (0.075 mg/m3), the amount of PM2.5 inhalated over 2 days is 0.0432 mg, and the concentration of PM2.5 exposure for each rat every 2 days is estimated to be 0.216 mg/kg body weight (bw). In the sampling period, the mean mass concentration of PM2.5 was 0.161 ± 0.060 mg/m3 on non-haze days,16 whereas on haze days, the PM2.5 concentration reached 0.692 ± 0.272 mg/m3.17 Herein, the average concentration of PM2.5 exposure for each rat every 2 days was estimated to range from 0.464 to 1.993 mg/kg bw. Besides this, the main PM2.5 doses were based on a report by Qiao et al.18 to explore the acute toxicological effects of PM2.5 on the lung as well as the mechanism of those effects. 2.4. Animal Experiments. Healthy adult, clean-grade male SD rats, weighing 180−200 g, were purchased from the Animal Center of Hebei Medical University. Animals were housed in metallic cages under standard conditions (24 ± 2 °C and 50 ± 5% humidity) with a 12 h light−dark cycle. Rats were divided randomly into five equal 409


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Chemical Research in Toxicology Table 1. Primer Sequences and PCR-Amplified Fragments Used in Real-Time RT-PCR genes

accession no.

OPA1 products Mfn1 products Mfn2 products Drp1 products Fis1 products actin products

NM_133585 107 bp NM_138976 143 bp NM_130894 136 bp NM_053655 120 bp NM_213746 162 bp NM_031144 211 bp

sequence forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer forward primer reverse primer

5′-CAGCTGGCAGAAGATCTCAAG-3′ 5′- CATGAGCAGGATTTTGACACC-3′ 5′-CCTTGTACATCGATTCCTGGGTTC-3′ 5′-CCTGGGCTGCATTATCTGGTG-3′ 5′- GATGTCACCACGGAGCTGGA-3′ 5′- AGAGACGCTCACTCACTTTG-3′ 5′-CGTAGTGGGAACTCAGAGCA-3′ 5′-TGGACCAGCTGCAGAATAAG-3′ 5′-AAATGATGCTACGCAGGCTT-3′ 5′- CCTGGACCATGACCAAGTTT-3′ 5′-CCTCTATGCCAACACAGTGC-3′ 5′-ATACTCCTGCTTGCTGATCC-3′

Figure 1. SEM images of lung tissues after intratracheal instillation in rats from the control (A), 1.5 mg/kg bw (B), 6.0 mg/kg bw (C), and 24 mg/kg bw (D) groups (deposited particles are indicated by red arrows); 15 000× magnification. 1:100 (for Mfn1 and Mfn2), or 1:1000 (for β-actin) in 1× PBS, 0.1% Tween, and 5% BSA overnight at 4 °C. The infrared-labeled anti-rabbit and anti-goat secondary antibodies (LI-COR Biosciences, USA), at a concentration of 1:5000, were added to nitrocellulose filter membranes and incubated for 1.5 h at room temperature. The membranes were scanned, and the band densities were quantified using the Odyssey Infrared Imaging System (Li-COR Biosciences, USA). 2.8. Measurement of MnSOD, MDA, Na+K+-ATPase, and Ca2+-ATPase Levels in Lung Mitochondria of Rats. Lung mitochondrial proteins were extracted with a protein extraction kit, and protein concentrations were detected with a protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Measurements of the enzymatic activity of MnSOD, Na+K+-ATPase, and Ca2+-ATPase as well as the content of MDA in lung mitochondria were performed spectrophotometrically using the corresponding kits from the Nanjing Jiancheng Biochemistry according to the manufacturer’s protocols.

of target gene expression was measured using the mRNA signal for the housekeeping gene actin as an internal control. The copy number of target gene/actin mRNA ratio was measured in all samples. The GenBank accession numbers and primer sequences of the tested genes and actin together with the length of the PCR-amplified fragments are listed in Table 1. 2.7. Western Blotting. Mitochondrial proteins from fresh lung tissues after the last exposure to PM2.5 were extracted with a mitochondria protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein concentrations were determined by a Bradford protein assay kit (Beyotime, Shanghai, China). Samples were mixed with loading buffer and boiled for 5 min. OPA1, Mfn1, Mfn2, Drp1, Fis1, and β-actin mitochondrial protein levels were detected by western blotting analysis as described previously.21 The rabbit polyclonal antibodies specific for rat Mfn1 and Mfn2 (Santa Cruz, CA, USA) and goat polyclonal antibodies specific for rat OPA1, Drp1, Fis1, and β-actin (Santa Cruz, CA, USA) were incubated at a concentration of 1:50 (for OPA1, Drp1, and Fis1), 410


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Figure 2. TEM images of lung tissues after intratracheal instillation in rats from the control (A), 1.5 mg/kg bw (B), 6.0 mg/kg bw (C), and 24 mg/ kg bw (D) groups (deposited particles are indicated by red arrows); 20 000× magnification.

Table 2. PM2.5 Relative Deposition and PM2.5-Induced Mitochondrial Damage in Lung Tissue of Rats in Different Groupsa itemb

control

PM deposition evaluation scoring using Flameng method linear regression analysis

− 0.21 ± 0.02

0.375 mg/kg PM2.5

1.5 mg/kg PM2.5

6.0 mg/kg PM2.5

∓ + ++ 0.23 ± 0.02 0.29 ± 0.02** 0.57 ± 0.06** Y (damage score) = 0.0229 × (dosage) + 0.2678; R2 = 0.87

24.0 mg/kg PM2.5 +++ 0.78 ± 0.02**

Derived from TEM results. For mitochondrial damage results, the values are mean ± SD from five individual samples. Using one-way ANOVA and in comparison with the control group, a significant difference is indicated by *, P < 0.05, and **, P < 0.01. bAbbreviations: −, no particles; ∓, very few particles; +, small amount of particles; ++, moderate amount of particles; +++, relatively more particles. a

in the presence of PM2.5 (10, 20, 50, 100, 200, 300, 500, 1000 μg/mL) was measured using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay, and the results were expressed as a percentage of the absorbance of control cells. The absorbance was read spectrophotometrically using a multifunctional microplate reader (Thermo Scientific Varioskan Flash, USA). On the basis of the MTT test results, the CL assay of the cells in the presence of PM2.5 or PM2.5 plus diphenyleneiodonium chloride (DPI) was performed as previous described.25 Briefly, 100 μL of a luminol solution and 100 μL of PM2.5 suspension (20, 50, 100, 200, 1000 μg/mL) was added to 1 mL of the cell suspension placed in a luminescence cuvette. After mixing, CL was recorded by a BPCL ultraweak luminescence analyzer (Beijing, China). Light intensity was monitored continuously by a recorder and expressed in counts per second (cps). The control cells were unstimulated macrophages exposed to PM2.5. Effects of the ROS generation inhibitor DPI (0.5 and 2.5 μM) on PM2.5-induced CL activity in AMs were investigated. Additionally, the effect of PM2.5 on luminol-dependent CL without cells was observed. 2.10. Statistical Analysis. Results were expressed as the mean ± standard deviation. Statistical analyses were performed by the use of one-way analysis of variance (ANOVA) with the SPSS19.0 package of programs for Windows. Posthoc tests were conducted to determine the difference between groups, followed by Fisher’s least significant difference (LSD) test. P < 0.05 was accepted as being statistically significant.

The sample suspension from the special control group treated with extracts of a blank filter did not induce pathological alterations or affect the levels of MnSOD, MDA, Na+K+-ATPase, and Ca2+-ATPase in lung mitochondria, and no statistical difference was observed between the normal control and special control groups (data not shown). 2.9. Rat AM Cell Culture and Chemiluminescence (CL) Measurements. AMs are the first defense against particles and microbes and play a critical role in the phagocytic removal of particles from the airways and alveoli. Also, AMs release ROS in response to encountering particles in a process referred to as the respiratory burst.22 ROS are generated from diverse sources. Compared to alveolar epithelial cells, AM-produced ROS is a primary and important way by which ROS are produced in the lung in response to both endogenous and exogenous stimuli.23 In this process, ROS and the subsequent release of inflammatory mediators may damage alveoli and alveolar epithelial cells, which are the downstream effectors of AMs. Therefore, AMs were used to measure the CL signals in response to PM2.5 in this study. The animals were anesthetized with sodium pentobarbital (80 mg/ kg bw), and AMs were obtained by tracheal lavage.24 Cells were centrifuged at 3000 rpm for 10 min at 4 °C and counted by trypan blue staining. AM cells were resuspended at a final concentration of 2.4 × 106 cells/mL in cold RPMI 1640 culture medium containing 10% fetal bovine serum and were incubated for 2 h at 37 °C with 5% CO2 and above 95% humidity to allow the AMs to adhere. Cell viability (%) 411


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Figure 3. Ultrastructural damage effects of PM2.5 exposure on lung and mitochondria of rats from the control (A), 0.375 mg/kg bw (B), 1.5 mg/kg bw (C), 6.0 mg/kg bw (D), and 24.0 mg/kg bw (E, F) groups; 20 000× magnification. The red arrows indicate sites of mitochondrial swelling, the blue arrow indicates site of mitochondrial cristae disorder or vacuolization, and the black arrows indicate sites of mitochondrial fission. AT-I, alveolar type I; AT-II, alveolar type II; N, nucleus; M, mitochondrion; LB, lamellar body; G, Golgi complex; L, lysosome; LD, lipid droplet; PM, particulate matter. In each panel, images on the right are high-magnification images corresponding to those on the left. AT I cell is a complex branched cell with multiple cytoplasmic plates that represents the gas-exchange surface in the alveolus, whereas AT II cell is cuboidal in shape and contains many organelles (such as mitochondria, lamellar bodies, lysosomes, and Golgi apparatus) that perform important biological functions.

3. RESULTS 3.1. Analysis of PM2.5 Chemical Characteristics. The chemical characteristics of PM2.5 used in this study were reported in our research.16,26 The levels of chrysene (CHR), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), benzo[a]anthracene (BaA), and benzo[g,h,i]perylene (BghiP), among 16 PAHs, in PM2.5 were obviously higher than the Chinese national standard for PAHs (10 ng/m3). The data from ion chromatography indicated that the daily mean level of SO42− and NO3− ions in the PM2.5 samples ranged from 4.89 to 5.87 μg/m3 and 1.69 to 1.71 μg/m3, respectively. 3.2. Observation of Particle Deposition in the Lung. PM2.5 deposition in the rat lung was observed in Figures 1 and 2, and its evaluation is in Table 2. The observation methods for PM2.5 deposition were similar to those of others.27,28 There were no particles in the rat lung in the control group (Figures

1A and 2A), whereas more particles were observed in the lung tissues in the presence of PM2.5 at concentrations of 1.5, 6.0, and 24.0 mg/kg bw (SEM image, Figure 1B−D; TEM image, Figure 2B−D). As observed in the SEM images, the particle sizes in the lungs were 532.8, 650.1, and 958.5 nm and 2.324 μm, less than 2.5 μm. The TEM results showed that PM mainly deposited in the alveoli and cytoplasm of the alveolar epithelia in the distal lung of rats. Moreover, the number of particles deposited in the lungs increased as the PM2.5 concentration increased (Table 2). This indicates that PM2.5 indeed deposited in the rat lungs after exposure to PM2.5 and that it was not eliminated from the lung. 3.3. Ultrastructural Damage Effects of PM2.5 on Lungs and Mitochondria. Ultrastructural examination is an effective tool to examine morphological changes and to evaluate the mitochondrial damaging effect of PM2.5 on lung alveolar epithelial cells in exposed rats. Generally, the alveolar epithelium of the lung is composed of two different cell 412


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Chemical Research in Toxicology types: alveolar type I (AT I) cells and alveolar type II (AT II) cells. AT I cells are thin and long and generally have few organelles in the cytoplasm, whereas AT II cells are characterized by a cytoplasm containing many mitochondria and osmiophilic multilamellar bodies. Comparisons of the ultrastructural changes in mitochondria in lung alveolar epithelial cells of rats in the different groups are depicted in Table 2 and Figure 3. The structures of AT I and AT II cells as well as mitochondria of the control group and the 0.375 mg/kg PM2.5 group were normal (Figure 3A,B). In the 1.5 mg/kg PM 2.5 -exposed group, some AT II cells had obvious pathological changes, including vacuolation of osmiophilic multilamellar bodies and mitochondrial swelling, and PM was found in the alveolar pneumocytes (Figure 3C). With the elevated dose of PM2.5, vacuolation of osmiophilic multilamellar bodies, mitochondrial swelling, disorder of cristae, mitochondrial vacuolation, some lysosomes, and irregularly compacted chromatin appeared in most of the AT II cells in the 6.0 mg/kg PM2.5-exposed rats (Figure 3D), whereas mitochondrial fission and a large number of lysosomes in AT I cells (Figure 3E) as well as mitochondrial membrane break, lipid droplets in the cytoplasm, and microvilli falling off in AT II cells (Figure 3F) were observed in the 24.0 mg/kg PM2.5-exposed rats. The results in Table 2 indicate that PM2.5 caused an increase in the mitochondrial damage scores in a concentration-dependent manner (R2 = 0.87). Such damage effects were significant in the presence of PM2.5 at the 1.5, 6.0, and 24.0 mg/kg bw doses relative to the control (P < 0.01). For example, swelling, cristae disorder, and membrane breach of the mitochondria markedly appeared with an increasing degree of pathological changes in the presence of PM2.5 (1.5 and 6.0 mg/kg bw). Mitochondrial membrane breach, cristae disorder, and fragmentation were more prominent in rats exposed to PM2.5 at a dose of 24.0 mg/ kg bw compared to that at a dose of 1.5 or 6.0 mg/kg bw. 3.4. Effects of PM2.5 on Rat Lung Mitochondrial Fusion/Fission Gene Expression. Changes in the levels of OPA1, Mfn1, Mfn2, Drp1, and Fis1 mRNA and protein in the lungs of rats exposed to PM2.5 are shown in Figure 4. PM2.5 at a concentration of 6.0 mg/kg bw significantly enhanced the OPA1 and Mfn1 mRNA and protein levels in rats compared with that in the control (P < 0.05), but PM2.5 at a concentration of 24.0 mg/kg bw significantly reduced the levels of OPA1 and Mfn1 compared to that of the control (P < 0.05 or P < 0.01). The 24.0 mg/kg bw PM2.5 group showed markedly increased Mfn2 mRNA and protein levels relative to that of the control (P < 0.05). No significant changes in the levels of OPA1, Mfn1, and Mfn2 in rats exposed to PM2.5 at concentrations of 0.375 and 1.5 mg/kg bw as well as in Mfn2 expression in the presence of 6.0 mg/kg bw PM2.5 were found. Conversely, Drp1 and Fis1 mRNA and protein levels showed an obvious increase in response to exposure of higher doses of PM2.5 (from 1.5 to 24 mg/kg bw) (P < 0.05 or P < 0.01). No significant transcriptional and translational differences in two mitochondrial fission genes were observed in rats in the presence of the lowest dose of PM2.5 (0.375 mg/kg bw). 3.5. Effects of PM2.5 on MnSOD, MDA, Na+K+-ATPase, and Ca2+-ATPase Levels in Lung Mitochondria of Rats. As shown in Figures 5 and 6, higher doses of PM2.5 (6.0 and 24.0 mg/kg bw) significantly inhibited the enzymatic activities of mitochondrial MnSOD, Na+K+-ATPase, and Ca2+-ATPase and obviously increased the MDA content in lung mitochondria compared with that in the control group (P < 0.05 or P < 0.01). No significant changes in the MnSOD and MDA levels

Figure 4. (A) mRNA expression of mitochondrial OPA1, Mfn1, Mfn2, Drp1, and Fis1 in the lung of rats treated with different concentrations of PM2.5; (B, C) protein levels of OPA1, Mfn1, Mfn2, Drp1, and Fis1 in lung mitochondria of rats treated with different concentrations of PM2.5. The control group was instilled with the same volume of physiological saline as that used to instill PM2.5 in the treated groups. Mean expression in each treated group is shown as increase/decrease compared to mean expression in the control group, which was ascribed an arbitrary value of 1. The values are the mean ± SD from five individual samples. Using one-way ANOVA and in comparison with the control group, significant difference is indicated by *, P < 0.05, and **, P < 0.01.

in rats exposed to PM2.5 at concentrations of 0.375 and 1.5 mg/ kg bw were observed. With the elevated doses of PM2.5, the MDA content increased in a dose-dependent manner (R2 = 413


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Figure 5. MnSOD activity and MDA content in lung mitochondria of rats treated with different concentrations of PM2.5. The control group was instilled with the same volume of physiological saline as that used to instill PM2.5 in the treated groups, and the other special control group (vehicle group) was instilled with the same volume of a suspension from extracts of a blank filter. The values are the mean ± SD from five individual samples. Using one-way ANOVA and in comparison with the control group, significant difference is indicated by *, P < 0.05, and **, P < 0.01.

Figure 6. Na +K +-ATPase and Ca 2+-ATPase activity in lung mitochondria of rats treated with different concentrations of PM2.5. The control group was instilled with the same volume of physiological saline as that used to instill PM2.5 in the treated groups, and the other special control group (vehicle group) was instilled with the same volume of a suspension from extracts of a blank filter. The values are the mean ± SD from five individual samples. Using one-way ANOVA and in comparison with the control group, significant difference is indicated by *, P < 0.05, and **, P < 0.01.

Figure 7. MTT test results (A), chemiluminescence of AMs (B) exposed to different doses of PM2.5, and inhibition of AM chemiluminescence by oxygen radical inhibitor DPI (0.5 and 2.5 μM) (C). Bars present the mean ± SD of three experiments. Significant difference is indicated by *, P < 0.05, and **, P < 0.01. Note that in panels B and C asterisks indicate effects significantly different from that in 0 μg dose cells (controls) compared to cells with PM2.5 or DPI.

0.67), implying that PM2.5 caused lipid peroxidation. In particular, PM2.5 at a dose of 1.5 mg/kg significantly decreased mitochondrial Na+K+-ATPase activity compared with that in the control group (P < 0.05). To assess the lipid peroxidation mechanism, the 20, 50, 100, 200, and 1000 μg/mL doses were chosen, on the basis of MTT assay results, to evaluate ROS production in AMs in response to PM2.5 (Figure 7). Cell viability was significantly reduced as the PM2.5 treatment doses increased from 50 to 1000 μg/mL PM2.5 relative to the control (Figure 7A). The results also showed that PM2.5 itself did not induce CL responses without cells. However, it caused an obviously acute CL response compared with the control cells (untreated cells with particles)

(P < 0.05 or P < 0.01), and CL of AMs reached a maximum value of about 116 cps when AMs were treated with 50 μg/mL of the PM2.5 suspension. This suggested that PM2.5 induced a respiratory burst in the AMs, accompanied by ROS release (Figure 7B). ROS generation inhibitor DPI, at 0.5 and 2.5 μM, caused a marked inhibition (47.0 and 84.1%) of AM CL responses (P < 0.05 or P < 0.01) (Figure 7C), which indicates 414


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mitochondrial fragmentation.36 On the contrary, Drp1 plays an important role in the mitochondrial fission process, whereas Fis1 participates in mitochondrial fission by recruiting cytoplasmic Drp1 into the mitochondrial outer membrane. Drp1 may punctuate spots on the mitochondrial surface to mediate mitochondrial fission for meeting energy needs.38 It appears that an elevated Drp1 level in mitochondria may be a good marker of mitochondrial fission. On the basis of the TEM image results (Figure 3C,D), mitochondrial swelling and fragmentation coexist in alveolar epithelial cells, whereas either mitochondrial swelling or fusion phenomena are significant relative to fission upon exposure to 1.5 and 6.0 mg/kg bw PM2.5. From the data shown in Figure 4, the expression of mitochondrial OPA1, Mfn1, Mfn2, Drp1, and Fis1 mRNA and protein was significantly enhanced at the 1.5 and/or 6.0 mg/kg bw PM2.5 doses compared with that in the controls. It may be speculated that, under PM2.5 exposure conditions (1.5 and/or 6.0 mg/kg bw), higher expression of OPA1 and Mfn1/2 in lung mitochondria is helpful for mitochondrial elongation, whereas overexpression of Drp1 and Fis1 can enhance mitochondrial fission to meet energy requirements. Furthermore, it is shown that dilation of the mitochondria with the cristae partly disordered and disappearing may induce damaging changes to lung mitochondria in rats exposed to higher doses of PM2.5. Interestingly, it was found that OPA1 and Mfn1 expression was markedly suppressed, whereas Drp1 and Fis1 expression was enhanced in the presence of PM2.5 at the highest dose (24.0 mg/kg bw). It is suggested that downregulation of OPA1 and Mfn1 expression and upregulation of Drp1 and Fis1 expression may promote the fission process, resulting in fragmented and scattered mitochondria. TEM images (Figure 3E,F) were in agreement with the above findings. More importantly, mitochondrial fragmentation may contribute to mitochondrial dysfunction, such as decreased ATP synthesis, increased ROS production, release of cytochrome C, and changes in Ca2+ signaling,37,39 resulting in cell damage, apoptosis, inflammation, dysfunction, and even cell death.40−42 Taken together, the abnormal alterations in mitochondrial morphology and the pattern of fusion−fission gene expression may provide an explanation for the possible mechanisms of PM2.5-induced lung damage. An in-depth study has shown that OPA1 cannot promote mitochondrial fusion in the absence of Mfn1 and that Mfn1 is unable to promote mitochondrial elongation if OPA1 is ablated.38 In the absence of Mfn2, the inner−outer membrane fusion machinery composed of OPA1 and Mfn1 is still intact and can provide a low degree of fusion.43 In this study, the tendency of OPA1 to be downregulated in the presence of PM2.5 at the highest dose (24.0 mg/kg bw) or for OPA1 to be upregulated at a dose of 6.0 mg/kg bw was consistent with Mfn1 expression, indicating that these two proteins might have a mutual function. We also paid attention to the relationships between lung mitochondria injury and ROS production and LPO. First, it has been demonstrated that PM2.5-induced oxidative stress is an important mechanism of PM2.5-mediated toxicity.44 PM2.5 may induce ROS primarily from site III of the mitochondrial electron transport chain,45 and in vitro experiments showed that PM2.5 may produce ROS, such as superoxide (O2•−) and hydroxyl (•OH) radicals.46 In this study, AMs released ROS in response to PM2.5 via a respiratory burst effect (Figure 7), which was consistent with the previous report.47 Macrophages contain NADPH oxidase in their cytoplasmic membranes,

that PM2.5 would be phagocytosed by AMs, an interaction that should trigger ROS release.

4. DISCUSSION Epidemiologic studies have demonstrated that the increased morbidity/mortality of pulmonary diseases is correlated with ambient PM2.5 pollution.7,8 PM2.5 is easily inhaled into the airway and deposited in lung alveoli, where the toxic particles may affect pulmonary structures and functions, triggering and aggravating pulmonary diseases.7,11,29 Clarifying the mechanisms of lung injury by ambient PM2.5 will be helpful to better understand the pathogenesis of respiratory system disease as a result of exposure to urban PM2.5 pollution. Therefore, we investigated the rat lung damaging effects of urban PM2.5 from five aspects, including morphological alterations in the lungs and mitochondria, the expression patterns of mitochondrial fusion and fission genes, ROS production, lipid peroxidation (LPO), and calcium homeostasis imbalance as well as the important chemical components in PM2.5 samples of Taiyuan winter. The morphological alterations in the lungs and mitochondria exposed to PM2.5 were observed first. In the lungs, AT I and AT II cells are crucial structural and functional cells, especially with regard to their contribution to epithelial repair.30 In other words, AT I and AT II injury induced by exogenous stimuli may lead to lung damage.31 As shown in the SEM and TEM results, PM2.5 deposited in the alveoli and cytoplasm of alveolar epithelia and caused pathological changes in AT I and AT II cells. For example, the disappearance of lamellar bodies and vacuolation in AT II cells as well as the presence of irregularly compacted chromatin in AT I and AT II cells after exposure to higher doses of PM2.5 were observed, whereas more lysosomes, lipid droplets, and microvilli falling off appeared in AT I and AT II cells in the presence of the highest dose of PM2.5. Lipid droplet formation in the cytoplasm reflects mitochondrial disruption and metabolic disturbance.32 The fragmentation and vacuolation of lamellar bodies may impair the normal functions of surfactants, influencing gas exchange.33 Under specific physiological or pathological conditions, the number of lysosomes may be increased to degrade some excessive endogenous or exogenous substances.34 When the lysosomal membrane is severely damaged, the hydrolytic enzymes from the lysosomes may enter the cytoplasm and digest the cell wall, resulting in cell injury.35 As for mitochondria, a higher dose of PM2.5 caused mitochondrial swelling and cristae disorder, whereas the highest dose of PM2.5 triggered mitochondrial fragment and cristae disappearance. Severe mitochondrial swelling and cristae disruption may result in mitochondrial membrane rupture and dysfunction.1,36 As a result, the morphological results suggest that particle deposition in the rat lung may cause severe structural and functional damage to the mitochondria or lung. Here, we specifically focused on and analyzed the correlation between lung mitochondrial damage and the expression patterns of mitochondrial fusion and fission genes in the lung in the presence of PM2.5. In mammalian cells, mitochondrial fusion is mediated by two mitofusins (Mfn1 and Mfn2) in the outer membrane and OPA1 in the inner membrane.37 Research has shown that overexpression of Mfn1/2 and OPA1 may promote fusion and the formation of the mitochondrial network, whereas a reduction in OPA1 and Mfn1 or Mfn2 may impair the fusion of mitochondrial membranes, resulting in incomplete fusion in a complex cell type or causing 415


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Chemical Research in Toxicology which generates large amounts of O2•− in response to phagocytosis.48 Among various inhibitors of oxygen radical generation, DPI targets the membrane NADPH oxidase complex and mitochondrial respiratory chain, and it may reduce mitochondrial superoxide production, probably through inhibiting NADH-ubiquinone oxidoreductase (complex I).49 It was found that the AM CL response to PM2.5 was significant compared with that of control cells without PM2.5 stimulation, and such an effect could be inhibited by about 84.1% with 2.5 μM DPI (Figure 7C). This observation indicates that PM2.5 may be intimately involved in the NADPH and mitochondrial respiratory chain dependent response and may enhance the ROS production of AMs by promoting a respiratory burst. Second, MnSOD is mainly expressed in the mitochondria, and it neutralizes a plethora of mitochondrial O2•−.50 MDA, the typical product of LPO, is often used as an index of LPO. Under stress conditions, the activity of antioxidative enzymes including SOD is inhibited. It is easy for excessive ROS to attack the mitochondrial membrane and form MDA, causing mitochondrial permeabilization, decreasing membrane potential, and promoting mitochondrial swelling.51 The overproduction of O2•− could account for the decrease in the OPA1 level and increase in the Drp1 level and could be an important mediator for mitochondrial fragmentation.52 PM2.5 at higher doses (6.0 and 24.0 mg/kg PM2.5) significantly suppressed MnSOD activity, increased ROS levels and MDA content, and affected OPA1 and Drp1 gene expression in lung mitochondria compared with the control group, accompanied by mitochondrial injury (Figures 3−5 and 7). It appears that high-dose-PM2.5-induced ROS accumulation and LPO, at least in part, explain the mechanism of mitochondrial damage. Third, it has been demonstrated that the induction of mitochondrial damage includes changes in membrane potential, generation of oxidative free radicals, uncoupling of oxidative phosphorylation, disequilibrium of calcium homeostasis, inhibition of the electron transport chain, etc.53,54 The ROS mechanism plays a very important role in mitochondrial damage, but it might be not the only way to damage mitochondria. The present data indicate that 1.5 mg/kg bw PM2.5 causes mitochondrial injury, as shown in Figure 3C, whereas it does not significantly change LPO, as shown in Figure 5. It is supposed that, to some extent, the mitochondrial injury effect induced by 1.5 mg/kg PM2.5 might be regulated in other ways. The ROS regulation mechanisms for PM-induced mitochondrial damage and fusion/fission gene expression need to be further studied comprehensively. Then, we studied the correlation between lung mitochondrial injury and calcium homeostasis imbalance. Na+K+-ATPase and Ca2+-ATPase, located in the mitochondrial membrane, can maintain the mitochondrial membrane electrochemical gradient and play a key role in ion homeostasis.55,56 Mitochondrial Na+K+-ATPase is a sodium pump that not only maintains a low concentration of sodium (Na+) and a high concentration of potassium (K+) inside the mitochondrion but also regulates the Ca2+ level in the mitochondrion.57 Mitochondrial Ca2+-ATPase is a calcium pump that can pump Ca2+ from the cytoplasm into the mitochondrion to maintain calcium homeostasis. ROS production and oxidative damage may inhibit Na+K+-ATPase and Ca2+-ATPase activity, which may trigger the loss of mitochondrial membrane potential and lead to Ca2+ overload.58,59 It is important to note that mitochondrial Ca2+ATPase activity inhibition blocks cytoplasmic Ca2+ from entering the mitochondria, elevating the levels of intracellular

Ca2+. In turn, mitochondrial membrane rupture, fluidity decrease, and permeability increase induced by LPO may promote the entrance of a high-level of intracellular Ca2+ into the mitochondria, inducing mitochondrial Ca2+ overload and further destroying mitochondrial structure and function.42 In addition, Na+K+-ATPase can reduce the mitochondrial Ca2+ concentration.57 In other words, decreased Na+K+-ATPase activity enhances mitochondrial Ca2+ overload. It has been demonstrated that ROS, Ca2+ overloading, decreased mitochondrial membrane potential, and mitochondrial permeability transition pore opening are the crucial causes of mitochondrial swelling, outer membrane rupture,60 or fragmentation under special conditions.61 As the results of Figure 6 show, PM2.5 significantly inhibited mitochondrial Na+K+-ATPase and Ca2+ATPase activity compared with that of the control group. Even exposure to PM2.5 at the lowest dose (0.375 mg/kg bw) decreased Na+K+-ATPase activity. This suggests that PM2.5 caused a decrease in Na+K+-ATPase and Ca2+-ATPase activity, leading to mitochondrial membrane injury and ion homeostatic imbalance, which may be one of the important causes of mitochondrial damage induced by PM2.5. In the present study, the PM2.5 samples were collected during the heating season from a representative of the coal-combustion polluted city, Taiyuan, and the PM chemical component analysis data showed that PAHs, sulfate, and nitrite in PM2.5 had reached high levels.16,26 What is the influence of PAHs, sulfate, and nitrite in PM2.5 on the lung injury? On one hand, PAHs have an important contribution to PM2.5-induced lung toxicity.62 Some studies have shown that exposure to PAHs is associated with mitochondrial damage via decreasing mitochondrial DNA (mtDNA) content, reducing mitochondrial membrane potential, and modulating the metabolic machinery inside the cells, accompanied by oxidative stress.63−65 This evidence suggests and important connection between PM2.5bound PAHs and lung damage or mitochondrial dysfunction. On the other hand, the epidemiologic and toxicological evidence provides little or no support for a causal association of PM sulfate or nitrate and health risk at ambient concentrations.66 However, there are some possible indirect processes through which sulfate and nitrate in PM may affect health-related end points, including interactions with certain metal species and a linkage with the production of secondary organic matter.66 Hence, it was hypothesized that adverse health effects observed after exposure to winter PM2.5 are partly caused by specific winter PM2.5 and PM components, i.e., PAHs. Since PM2.5 is a complex mixture of organic and inorganic components that include metals, salts, PAHs, and carbonaceous material, further work may be needed to investigate the toxicological roles of the components of PM2.5 on mitochondrial damage in respiratory diseases.

5. CONCLUSIONS The results from the present research showed that (1) PM2.5bound PAHs, sulfate, and nitrate during the winter inTaiyuan reached higher levels; (2) PM2.5 could be deposited in the pulmonary alveoli of rats and cause abnormal ultrastructural changes to the mitochondria and lung; (3) PM2.5 induced mitochondrial expression of OPA1, Mfn1, Mfn2, Drp1, and Fis1 mRNA and protein; and (4) PM2.5 produced ROS and that higher doses of PM2.5 induced LPO and decreased Na+K+ATPase and Ca2+-ATPase activity. The data support the hypothesis that higher doses of PM2.5 may induce lung mitochondrial damage, probably through the imbalance of 416


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Chemical Research in Toxicology fusion and fission gene expression, ROS and LPO formation, and ion homeostatic imbalance, hence elevating the possibility of triggering lung diseases under certain PM2.5 pollution conditions.

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damage to lung tissue and peripheral blood in endotracheal PM2.5treated rats. Biomed. Environ. Sci. 22, 223−228. (10) Riva, D. R., Magalhães, C. B., Lopes, A. A., Lanças, T., Mauad, T., Malm, O., Valença, S. S., Saldiva, P. H., Faffe, D. S., and Zin, W. A. (2011) Low dose of fine particulate matter (PM2.5) can induce acute oxidative stress, inflammation and pulmonary impairment in healthy mice. Inhalation Toxicol. 23, 257−267. (11) Upadhyay, D., Panduri, V., Ghio, A., and Kamp, D. W. (2003) Particulate matter induces alveolar epithelial cell DNA damage and apoptosis: role of free radicals and the mitochondria. Am. J. Respir. Cell Mol. Biol. 29, 180−187. (12) Li, N., Sioutas, C., Cho, A., Schmitz, D., Misra, C., Sempf, J., Wang, M., Oberley, T., Froines, J., and Nel, A. (2003) Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ. Health Perspect. 111, 455−460. (13) Meyer, J. N., Leung, M. C., Rooney, J. P., Sendoel, A., Hengartner, M. O., Kisby, G. E., and Bess, A. S. (2013) Mitochondria as a target of environmental toxicants. Toxicol. Sci. 134, 1−17. (14) Hou, L., Zhu, Z. Z., Zhang, X., Nordio, F., Bonzini, M., Schwartz, J., Hoxha, M., Dioni, L., Marinelli, B., Pegoraro, V., Apostoli, P., Bertazzi, P. A., and Baccarelli, A. (2010) Airborne particulate matter and mitochondrial damage: a cross-sectional study. Environ. Health 9, 48. (15) Xia, T., Kovochich, M., and Nel, A. E. (2007) Impairment of mitochondrial function by particulate matter (PM) and their toxic components: implications for PM-induced cardiovascular and lung disease. Front. Biosci. 12, 1238−1246. (16) Li, R., Kou, X., Geng, H., Dong, C., and Cai, Z. (2014) Pollution characteristics of ambient PM2.5-bound PAHs and NPAHs during winter time in Taiyuan. Chin. Chem. Lett. 25, 663−666. (17) Cao, L., Geng, H., Yao, C., Zhao, L., Duan, P., Xuan, Y., and Li, H. (2014) Investigation of chemical compositions of atmospheric fine particles during a wintertime haze episode in Taiyuan City. China Environ. Sci. 34, 259−265. (18) Qiao, G., Zhang, Z., Dong, J., Qiu, Y., Guo, L., Bai, Y., Song, J., and Chen, G. (2011) The effect of subacute PM2.5 exposure related to traffic on cytokine levels in BALF and serum of rats. J. Toxicol. 25, 275−277. (19) Bai, R., Zhang, L., Liu, Y., Meng, L., Wang, L., Wu, Y., Li, W., Ge, C., Le Guyader, L., and Chen, C. (2010) Pulmonary responses to printer toner particles in mice after intratracheal instillation. Toxicol. Lett. 199, 288−300. (20) Flameng, W., Borgers, M., Daenen, W., and Stalpaert, G. (1980) Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J. Thorac. Cardiovasc. Surg. 79, 413− 424. (21) Li, R., Meng, Z., and Xie, J. (2007) Effects of sulfur dioxide derivatives on four asthma-related gene expressions in human bronchial epithelial cells. Toxicol. Lett. 175, 71−81. (22) Beck-Speier, I., Dayal, N., Karg, E., Maier, K. L., Schumann, G., Schulz, H., Semmler, M., Takenaka, S., Stettmaier, K., Bors, W., Ghio, A., Samet, J. M., and Heyder, J. (2005) Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles. Free Radical Biol. Med. 38, 1080−1092. (23) Chow, C. W., Herrera Abreu, M. T., Suzuki, T., and Downey, G. P. (2003) Oxidative stress and acute lung injury. Am. J. Respir. Cell Mol. Biol. 29, 427−431. (24) Geng, H., Meng, Z., and Zhang, Q. (2006) In vitro responses of rat alveolar macrophages to particle suspensions and water-soluble components of dust storm PM2.5. Toxicol. In Vitro 20, 575−584. (25) Geng, H., and Meng, Z. (2006) Inhibition of superoxide dismutase, vitamin C and glutathione on chemiluminescence produced by luminol and the mixture of sulfite and bisulfite. Spectrochim. Acta, Part A 64, 87−92. (26) Li, R., Geng, H., Cai, Z., Song, Z., Xu, L., Chen, J., and Chen, M. (2014) Pollution characteristics of PM2.5 PAHs, NPAHs, SO42−and NO3− in wintertime in Taiyuan city. Res. Appl. Build. Mater. 177, 13− 15.

AUTHOR INFORMATION

Corresponding Authors

*(Z.C.) Tel: 00852-34117070; Fax: 00852-34117348; E-mail: [email protected]. *(C.D.) Tel/Fax: 0086-351-7011011; E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (nos. 21177078, 21175086, and 21175025), a research project supported by Shanxi Scholarship Council of China (2013-16), the Nature Science Foundation of Shanxi Province in China (2014011036-2), and the 100 Talents Program of Shanxi Province. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AT I, alveolar type I; AT II, alveolar type II; AMs, alveolar macrophages; CL, chemiluminescence; DPI, diphenyleneiodonium chloride; Drp1, dynamin-related protein 1; PM2.5, fine particulate matter; Fis1, fission-mediator protein 1; OH, hydroxyl radical; LPO, lipid peroxidation; MDA, malondialdehyde; Mfn, mitofusin; MnSOD, Mn superoxide dismutase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OPA1, optic atrophy protein 1; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate buffer solution; QFFs, quartz fiber filters; ROS, reactive oxygen species; SEM, scanning electron microscope; O2•−, superoxide radical; TEM, transmission electron microscope

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REFERENCES

(1) Hoppins, S. (2014) The regulation of mitochondrial dynamics. Curr. Opin. Cell Biol. 29C, 46−52. (2) Mabalirajan, U., Dinda, A. K., Kumar, S., Roshan, R., Gupta, P., Sharma, S. K., and Ghosh, B. (2008) Mitochondrial structural changes and dysfunction are associated with experimental allergic asthma. J. Immunol. 181, 3540−3548. (3) Sureshbabu, A., and Bhandari, V. (2013) Targeting mitochondrial dysfunction in lung diseases: emphasis on mitophagy. Front. Physiol. 4, 384. (4) Westrate, L. M., Drocco, J. A., Martin, K. R., Hlavacek, W. S., and Mackeigan, J. P. (2014) Mitochondrial morphological features are associated with fission and fusion events. PLoS One 9, e95265. (5) Cao, J. J., Shen, Z. X., Chow, J. C., Watson, J. G., Lee, S. C., Tie, X. X., Ho, K. F., Wang, G. H., and Han, Y. M. (2012) Winter and summer PM2.5 chemical compositions in fourteen Chinese cities. J. Air Waste Manage. Assoc. 62, 1214−1226. (6) Li, N., Hao, M., Phalen, R. F., Hinds, W. C., and Nel, A. E. (2003) Particulate air pollutants and asthma. A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin. Immunol. 109, 250−265. (7) Li, P., Xin, J., Wang, Y., Wang, S., Li, G., Pan, X., Liu, Z., and Wang, L. (2013) The acute effects of fine particles on respiratory mortality and morbidity in Beijing, 2004−2009. Environ. Sci. Pollut. Res. Int. 20, 6433−6444. (8) Vinikoor-Imler, L. C., Davis, J. A., and Luben, T. J. (2011) An ecologic analysis of county-level PM2.5 concentrations and lung cancer incidence and mortality. Int. J. Environ. Res. Public Health 8, 1865− 1871. (9) Lin, Z. Q., Xi, Z. G., Yang, D. F., Chao, F. H., Zhang, H. S., Zhang, W., Liu, H. L., Yang, Z. M., and Sun, R. B. (2009) Oxidative 417


Article


mitochondrial reactive oxygen species production. Biochem. Biophys. Res. Commun. 253, 295−299. (50) Miriyala, S., Holley, A. K., and St. Clair, D. K. (2011) Mitochondrial superoxide dismutasesignals of distinction. Adv. Anticancer Agents Med. Chem. 11, 181−190. (51) Castilho, R. F., Meinicke, A. R., Almeida, A. M., Hermes-Lima, M., and Vercesi, A. E. (1994) Oxidative damage of mitochondria induced by Fe(II) citrate is potentiated by Ca2+ and includes lipid peroxidation and alterations in membrane proteins. Arch. Biochem. Biophys. 308, 158−163. (52) Makino, A., Scott, B. T., and Dillmann, W. H. (2010) Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes. Diabetologia 53, 1783−1794. (53) Rousset, S., Alves-Guerra, M. C., Mozo, J., Miroux, B., CassardDoulcier, A. M., Bouillaud, F., and Ricquier, D. (2004) The biology of mitochondrial uncoupling proteins. Diabetes 53, S130−S135. (54) Slimen, I. B., Najar, T., Ghram, A., Dabbebi, H., Ben Mrad, M., and Abdrabbah, M. (2014) Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. Int. J. Hyperthermia 30, 513− 523. (55) Artigas, P., and Gadsby, D. C. (2002) Ion channel-like properties of the Na+/K+ Pump. Ann. N.Y. Acad. Sci. 976, 31−40. (56) de Meis, L., Arruda, A. P., da Costa, R. M., and Benchimol, M. (2006) Identification of a Ca2+-ATPase in brown adipose tissue mitochondria: regulation of thermogenesis by ATP and Ca2+. J. Biol. Chem. 281, 16384−16390. (57) Motegi, K., Tanonaka, K., Takenaga, Y., Takagi, N., and Takeo, S. (2007) Preservation of mitochondrial function may contribute to cardioprotective effects of Na +/Ca2+ exchanger inhibitors in ischaemic/reperfused rat hearts. Br. J. Pharmacol. 151, 963−978. (58) Golod, E. A. (1996) Lipid peroxidation and Ca-dependent ATPase activity in the microsomal fraction of renal tissue in patients with nephrolithiasis and chronic pyelonephritis. Urol. Nefrol. 5, 14−16. (59) Thévenod, F., and Friedmann, J. M. (1999) Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K+-ATPase through proteasomal and endo-/lysosomal proteolytic pathways. FASEB J. 13, 1751−1761. (60) Rodriguez-Enriquez, S., He, L., and Lemasters, J. J. (2004) Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int. J. Biochem. Cell Biol. 36, 2463−2472. (61) Watanabe, T., Saotome, M., Nobuhara, M., Sakamoto, A., Urushida, T., Katoh, H., Satoh, H., Funaki, M., and Hayashi, H. (2014) Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance. Exp. Cell Res. 323, 314−325. (62) Abbas, I., Saint-Georges, F., Billet, S., Verdin, A., Mulliez, P., Shirali, P., and Garçon, G. (2009) Air pollution particulate matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model. Toxicol. In Vitro 23, 37−46. (63) Pavanello, S., Dioni, L., Hoxha, M., Fedeli, U., MielzynskaSvach, D., and Baccarelli, A. A. (2013) Mitochondrial DNA copy number and exposure to polycyclic aromatic hydrocarbons. Cancer Epidemiol., Biomarkers Prev. 22, 1722−1729. (64) Gurbani, D., Bharti, S. K., Kumar, A., Pandey, A. K., Ana, G. R., Verma, A., Khan, A. H., Patel, D. K., Mudiam, M. K., Jain, S., Roy, R., and Dhawan, A. (2013) Polycyclic aromatic hydrocarbons and their quinones modulate the metabolic profile and induce DNA damage in human alveolar and bronchiolar cells. Int. J. Hyg. Environ. Health 216, 553−565. (65) Jiang, Y., Zhou, X., Chen, X., Yang, G., Wang, Q., Rao, K., Xiong, W., and Yuan, J. (2011) Benzo(a)pyrene-induced mitochondrial dysfunction and cell death in p53-null Hep3B cells. Mutat. Res. 726, 75−83. (66) Reiss, R., Anderson, E. L., Cross, C. E., Hidy, G., Hoel, D., McClellan, R., and Moolgavkar, S. (2007) Evidence of health impacts of sulfate- and nitrate-containing particles in ambient air. Inhalation Toxicol. 19, 419−449.

(27) Geiser, M., and Kreyling, W. G. (2010) Deposition and biokinetics of inhaled nanoparticles. Part. Fibre Toxicol. 7, 2. (28) Herzog, F., Clift, M. J., Piccapietra, F., Behra, R., Schmid, O., Petri-Fink, A., and Rothen-Rutishauser, B. (2013) Exposure of silvernanoparticles and silver-ions to lung cells in vitro at the air−liquid interface. Part. Fibre Toxicol. 10, 11. (29) You, Y., and Bai, Z. (2012) Research advances in exposure to ambient particulate matter and health effects. Asian J. Ecotoxicol. 7, 123−132. (30) Fehrenbach, H. (2001) Alveolar epithelial type II cell: defender of the alveolus revisited. Respir. Res. 2, 33−46. (31) Ward, H. E., and Nicholas, T. E. (1984) Alveolar type I and type II cells. Aust. N. Z. J. Med. 14, 731−734. (32) Lee, S. J., Zhang, J., Choi, A. M., and Kim, H. P. (2013) Mitochondrial dysfunction induces formation of lipid droplets as a generalized response to stress. Oxid. Med. Cell. Longevity 2013, 327167. (33) Schmitz, G., and Müller, G. (1991) Structure and function of lamellar bodies, lipid−protein complexes involved in storage and secretion of cellular lipids. J. Lipid Res. 32, 1539−1570. (34) Luzio, J. P., Pryor, P. R., and Bright, N. A. (2007) Lysosomes: fusion and function. Nat. Rev. Mol. Cell Biol. 8, 622−632. (35) Boya, P. (2012) Lysosomal function and dysfunction: mechanism and disease. Antioxid. Redox Signaing 17, 766−774. (36) Griparic, L., van der Wel, N. N., Orozco, I. J., Peters, P. J., and van der Bliek, A. M. (2004) Loss of the intermembrane space protein Mgm1/OPA1 induces swelling and localized constrictions along the lengths of mitochondria. J. Biol. Chem. 279, 18792−18798. (37) Youle, R. J., and van der Bliek, A. M. (2012) Mitochondrial fission, fusion, and stress. Science 337, 1062−1065. (38) Smirnova, E., Shurland, D. L., Ryazantsev, S. N., and van der Bliek, A. M. (1998) A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143, 351−358. (39) Chan, D. C. (2012) Fusion and fission: interlinked processes critical for mitochondrial health. Annu. Rev. Genet. 46, 265−287. (40) Guo, Y., Zhang, W., Yan, Y. Y., Ma, C. G., Wang, X., Wang, C., and Zhao, J. L. (2013) Triterpenoid pristimerin induced HepG2 cells apoptosis through ROS-mediated mitochondrial dysfunction. J. BUON 18, 477−485. (41) Kaddour-Djebbar, I., Choudhary, V., Brooks, C., Ghazaly, T., Lakshmikanthan, V., Dong, Z., and Kumar, M. V. (2010) Specific mitochondrial calcium overload induces mitochondrial fission in prostate cancer cells. J. Oncol. 36, 1437−1444. (42) López-Armada, M. J., Riveiro-Naveira, R. R., Vaamonde-García, C., and Valcárcel-Ares, M. N. (2013) Mitochondrial dysfunction and the inflammatory response. Mitochondrion 13, 106−118. (43) Cipolat, S., Martins de Brito, O., Dal Zilio, B., and Scorrano, L. (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A. 101, 15927−15932. (44) Dellinger, B., Pryor, W. A., Cueto, R., Squadrito, G. L., Hegde, V., and Deutsch, W. A. (2001) Role of free radicals in the toxicity of airborne fine particulate matter. Chem. Res. Toxicol. 14, 1371−1377. (45) 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., and Budinger, G. R. (2009) Mitochondrial complex III-generated oxidants activate ASK1 and JNK to induce alveolar epithelial cell death following exposure to particulate matter air pollution. J. Biol. Chem. 284, 2176−2186. (46) Sagai, M., Saito, H., Ichinose, T., Kodama, M., and Mori, Y. (1993) Biological effects of diesel exhaust particles. I. In vitro production of superoxide and in vivo toxicity in mouse. Free Radical Biol. Med. 14, 37−47. (47) Miles, P. R., Castranova, V., and Lee, P. (1978) Reactive forms of oxygen and chemiluminescence in phagocytizing rabbit alveolar macrophages. Am. J. Physiol. 235, 103−108. (48) Iles, K. E., and Forman, H. J. (2002) Macrophage signaling and respiratory burst. Immunol. Res. 26, 95−105. (49) Li, Y., and Trush, M. A. (1998) Diphenyleneiodonium, an inhibitor of NAD(P)H oxidase inhibitor, also potently inhibits 418


Effect of Ambient PM2.5 on Lung Mitochondrial Damage and Fusion

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