Seasonal Variation in Air Particulate Matter (PM10) Exposure-Induced

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Seasonal Variation in Air Particulate Matter (PM10) Exposure-Induced Ischemia-Like Injuries in the Rat Brain Lin Guo, Ben Li, Juan-juan Miao, Yang Yun, Guang-ke Li, and Nan Sang* College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, P. R. China ABSTRACT: Epidemiological studies imply a significantly positive association between particulate matter (PM) level and ischemic stroke hospitalization. However, considering that PM10 is highly heterogeneous and varies with season within the same location, existing experimental evidence remained low. In the present study, we first treated Wistar rats with PM10 samples collected from different seasons in Taiyuan, a typically coal-burning city of China, and determined ischemia-related markers in the cortex. The results indicated that PM10 exposure caused endothelial dysfunction, inflammatory response, and neuro-functional impairment similar to that of cerebral ischemia with season-dependent properties, and the winter sample presented the most obvious injuries. Then, we detected the chemical composition of PM10 samples followed by analysis of their correlation with the above biomarkers and found that winter PM10, characterized by higher polycyclic aromatic hydrocarbons (PAHs) and carbon load, played the major role in causing brain ischemia-like injuries among different season samples. Furthermore, by setting up an ischemic neuron model in vitro, we confirmed that winter PM10 presented the most serious aggravation on ischemia-produced injury outcome. This study provides experimental evidence for clarifying the association between season-dependent PM10 pollution in the atmospheric environment and an increased risk of ischemia-like injuries.

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INTRODUCTION In addition to the extensive literature documenting the association between air pollution and respiratory and cardiovascular diseases, recent evidence shows a link between elevated short-term outdoor air pollution and an increased risk of cerebrovascular disorders. Globally, ambient air pollution caused 3.7 million deaths in 2012, and the five diseases contributing to deaths were ischemic heart disease (IHD) (40%), stroke (40%), chronic obstructive pulmonary disease (COPD) (11%), lung cancer (6%), and acute lower respiratory infection (ALRI) (3%).1 In particular, epidemiological studies show a significantly positive association between particulate matter levels and other gaseous pollutants and hospitalization for ischemic stroke events in a number of cities.2−7 A study of Edmonton, Canada presented that an interquartile increase with same day exposures to SO2, NO2, CO, and PM10 was associated with an increased risk of an emergency department visit for ischemic stroke.3,8 Similarly, an analysis of nine U.S. cities indicates that an interquartile range increase in PM10, CO, NO2, and SO2 exposure consistented of an increased risk of hospital admission for ischemic stroke.9 The data from Como urban area citizens of Italy shows that NO2 and PM10 were significantly associated with admission and mortality and with estimated relative risk (RR) of 1.039 (95% confidence interval [CI] 1.066−1.013) and 1.078 (95% CI 1.104−1.052) for hospital admission at 2- and 4-day lag times, respectively.10 Following analysis based on 1680 consecutive patients admitted to Mantua (Italy) Stroke Unit indicates that air pollution peaks © XXXX American Chemical Society

contributed to an increased risk of hospitalization for stroke and that particulate matter might be a significant risk factor.11 As one of the major causes of death and frequent disease leading to disability, ischemic stroke affects up to 0.2% of the population every year with a high incidence.12 In China, the rate of ischemic stroke increases by almost 9% every year.13 Therefore, more attention has been paid to define the presence of effects and the type of pollutants most responsible using experimental studies. While several air pollutants have been studied for their links to ischemic stroke hospital admission and mortality, particulate matter (PM10) has emerged as a pollutant of interest. Convincing evidence indicates that PM10 (particles ≤10 μm in aerodynamic diameter, including fine and ultrafine particles) causes the most serious effects on human health because of a broad range of toxic compounds presenting in this PM fraction,14,15 such as transition metals, endotoxins,16 and ultrafine components. However, urban PM10 is highly heterogeneous and varies with season and within and between cities.17,18 As reported, polycyclic aromatic hydrocarbons (PAHs), an organic class of PM components, differ in PM concentration between summer and winter seasons.19−21 Therefore, seasonal variation of PM10 composition is related to different bioresponses, and ischemic stroke might be one of the disorders affected.22 Special Issue: Chemical Toxicology in China Received: September 26, 2014

A

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology Table 1. Sequences of Primers and Cycling Conditions temperature (t) gene

accession no.

sequence

β-actin

NM_031144

ET-1

NM_012548

eNOS

NM_021838

IL-1 β

NM_031512

COX-2

NC_001665.2

iNOS

NM_012967

ICAM-1

NM_012967

sense: 5′-GCCCTAGACTTCGAGCAAGAG-3′ antisense: 5′-AGCACTGTGTTGGCATAGAGGT-3′ sense: 5′-AAGCGATCCTTGAAAGACTTACTTC-3′ antisense: 5′TGTGTATCAACTTCTGGTCTCTGTA-3′ sense: 5′-CAGCGCCCACCCAGGAGAG-3′ antisense: 5′-ATCGGCAGCCAAACACCAAAGTC-3′ sense: 5′-GCCTCAAGGGGAAGAATCTATACC-3′ antisense: 5′-GGGAACTGTGCAGACTCAAACT-3′ sense: 5′-AAATCGGGAGTTGGAATCACTTTC-3′ antisense: 5′-CCATCGTTTAGGACAGAACATCAC3′ sense: 5′-CAGAAGCAGAATGTGACCATCAT-3′ antisense: 5′-CGGAGGGACCAGCCAAATC-3′ sense: 5′-TTCAACCCGTGCCAGGC-3′ antisense: 5′-GTTCGTCTTTCATCCAGTTAGTCT3′

denaturation

annealing

extended

cycles

95 °C (3 min)

94 °C (20 s)

55 °C (20 s)

72 °C (20 s)

55

95 °C (3 min)

94 °C (20 s)

55 °C (20 s)

72 °C (20 s)

55

95 °C (3 min)

94 °C (20 s)

56 °C (20 s)

72 °C (20 s)

55

95 °C (3 min)

94 °C (20 s)

55 °C (20 s)

72 °C (20 s)

55

95 °C (3 min)

94 °C (20 s)

55 °C (20 s)

72 °C (20 s)

55

95 °C (3 min)

94 °C (20 s)

55 °C (20 s)

72 °C (20 s)

55

95 °C(3 min)

94 °C (20 s)

58 °C (20 s)

72 °C (20 s)

55

rate of the sampler was 100 L/min, and the sampling time was nominally 24 h/d. PM10 mass concentration in ambient air was determined by the gravimetric method.26 The contents of inorganic ions, metals, and elements, and PAHs were analyzed by ion chromatography, inductively coupled plasma-mass spectrometry or GC-MS, respectively. The method of sample collection references the previous study of our laboratory.27 Animals and PM10 Exposure. Male Wistar rats weighing 190− 210 g, were purchased from the Experimental Animal Center, Academy of Military Medical Sciences of Chinese PLA (Beijing, China). After normally screened for common rat pathogens, the rats were raised in specific clean cages with hardwood chip bedding under standard conditions of a 12-h light/dark cycle, and sufficient food and water were provided. The method of intratracheal instillation references the previous study of our laboratory.27 The rats were randomly divided into five equal groups of six animals each, and the experiment was carried out in duplicate. The animals in four treatment groups were anesthetized by chloral hydrate and then intratracheally instilled with a PM10 suspension obtained during different seasons to a final exposure concentration of 10 mg/kg bw. The control group was instilled with the same amount of physiological saline. Instillation was performed five times for 3 days each. The animals were sacrificed by decapitation 48 h after the last exposure, and the cortex was stripped from the brain. After quick freezing in liquid nitrogen, the cortex was stored at −80 °C. The care and use of the animals reported in this study were approved by the Institutional Animal Care and Use Committee of Shanxi University. Oxygen and Glucose Deprivation (OGD) Model and PM10 Treatment. The rat cortex was dissected and incubated in oxygenated trypsin for 10 min at 37 °C and then mechanically triturated. The cells were spun down and resuspended in Neurobasal/B27 medium (Invitrogen, Carlsbad, CA) supplemented with 0.5 mM L-glutamine, penicillin/streptomycin, and 25 μM glutamate. The cells (1 × 106) were loaded into poly-D-lysine-coated 35 mm culture dishes for subsequent assay. To establish the OGD model, the culture media were replaced by prewarmed DMEM with low glucose (1 g/L), and the cultures were then transferred to an anaerobic chamber (Thermo Forma Scientific, USA) that was filled with an anoxic gas mixture (5% CO2 and 95% N2) at 37 °C for 45 min and recovered for another 24 h under normal conditions. OGD was terminated by returning the cultures to the normoxic incubator with normal culture medium.28−30 Following this, the model cultures were randomly divided into five equal groups: four treatment groups were individually treated with 100 μg/mL PM10 from different seasons for 24 h, and the vehicle group

The cerebrovascular endothelium, marked by endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS), plays a critical role in the regulation of normal vascular homeostasis, and perturbation of endothelial function causes blood flow reduction, energy metabolism interruption, and subsequent injuries associated with ischemia in the brain.23,24 Also, focal cerebral ischemia is related with a marked inflammatory reaction and neuronal apoptosis. Overproduction of enzymes (inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)), cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (Il-1β)), and adhesion molecules (intercellular adhesion molecule 1 (ICAM-1)) increases blood coagulation and promotes thrombus formation, and then leads to disturbance in the blood supply to the brain, eventually contributing to both necrotic and apoptotic neuronal death.25 Our present study aimed to clarify an association between season-dependent PM10 pollution and the enhanced risk of cerebral ischemia-like injuries. First, we treated Wistar rats with PM10 samples collected from different seasons in Taiyuan, a typically coal-burning city of China, and determined the seasondependent changes of cerebral ischemia-related markers in the cortex, including endothelial function (ET-1, eNOS), inflammatory marker level (iNOS, COX-2, Il-1β, and ICAM-1), and subsequent neuro-functional impairment. Following this, we analyzed the chemical composition of PM10 samples and calculated their correlative coefficient with the above biomarkers, and clarified that winter PM10, mainly characterized by the highest PAHs and carbon levels, played the major role in aggravating cerebral ischemia-like injuries among different season samples.

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initial denaturation

MATERIALS AND METHODS

PM10 Sampling, Chemical Analysis, and Suspension Preparation. The samples were collected from Shanxi University (112°57′E longitude, 37°73′N latitude), Taiyuan City, Shanxi Province of China. No specific permissions were required for the location, and the field studies did not involve endangered or protected species. PM10 samples were collected on glass filters year-round starting March 15, 2011 and including four seasons (every three months represents one season) by a PM10 middle volume air sampler (TH-150C, Wuhan, China), which was placed on the top of the building far away from obstacles so as to get free-moving air. The flow B

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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was treated with the same amount of 0.9% physiological saline. Each group was divided into three subgroups. Immunofluorescence and TUNEL Staining. The tissue was rapidly removed, washed for several times with 0.1 M phosphate buffer saline (PBS, pH 7.4), fixed in 10% formalin for 24 h at room temperature, dehydrated by graded ethanol, and embedded in paraffin. Sections (5−6-μm-thick) were deparaffinized with xylene, and antigen was retrieved by citric acid buffer (pH 6.0). Then, the sections were blocked with 3% BSA solution for 30 min at room temperature and incubated with the anti-ET-1 primary antibody overnight at 4 °C and the corresponding antirabbit secondary antibodies for 1 h at room temperature. After staining with DAPI (4′,6-diamidino-2-phenylindole), a fluorescent stain which binds strongly to DNA, the sections were mounted, coverslipped, and stored in darkness at 4 °C until images were taken. For TUNEL staining, sections (5−6-μm-thick) were deparaffinized with xylene and then processed for the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay according to the manufacturer’s instructions (Apoptag Plus Peroxidase in situ Apoptosis Detection Kit, Roche Company, Purchase, Shanghai). Transmission Electron Microscope (TEM) Observation. About 1 mm × 1 mm × 1 mm pieces were rapidly cut from the cortex in the brain and fixed in stationary liquid (2 h) and buffer solution (overnight). After that, they were en bloc stained with 2% aqueous uranyl acetate for 1 h at room temperature in the dark, dehydrated by graded ethanol, and embedded in beam capsules. Sections, 70−80 nmthick, were cut from the embedded tissue and collected onto grids to air-dry overnight. The grids were stained with uranyl acetate for 15−30 min and lead citrate for 3−15 min, and then the synaptic structural parameters were observed and measured by a transmission electron microscope and image analysis instrument (JEOL, JEM 1400). Reverse Transcription and Real-Time Reverse Transcription (RT)-PCR Analysis. Total RNA was extracted by TRIzol Reagent (Invitrogen Life Technologies) and quantified by optical density at 260 nm. First-strand complementary DNA (cDNA) was synthesized using the reverse transcription kit (TaKaRa Biotechnology Co., Ltd., Dalian) and stored at −20 °C until use. Each PCR reaction contained cDNA, primers, and SYBR-Green. The primers sequences and cycling conditions are listed in Table 1. Reactions were run on a Bio-Rad iQ5 Real-Time Cycler (Corbett Research, Sydney, Australia). The relative quantification of the expression of the target genes was measured using β-actin mRNA as an internal control. Protein Isolation and Immunoblot Analysis. Total protein was isolated by lysis buffer and quantified by bicinchoninic acid assay, then 50 μg of total proteins was separated by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the membranes were incubated in rabbit polyclonal antibodies ET-1, eNOS, iNOS, ICAM-1 (Beijing Boisynthesis Biotechnology CO., LTD), rabbit polyclonal antibody for COX-2 (Cayman Chemical, USA), or rabbit monoclonal antibody for β-actin and rabbit polyclonal antibody for cleaved caspase-3 (Cell Signaling, USA) at a concentration of 1:100 (for ET-1, eNOS, iNOS, and ICAM-1), 1:200 (for COX-2), or 1:5000 (for β-actin or cleaved caspase-3) at 4 °C overnight. Exposure to fluorescently labeled secondary antibody (1:2000) (IRDye 800CW Goat anti-Rabbit IgG (H + L), LI-COR) was followed by scanning and detecting with a LI-COR Odyssey infrared fluorescence instrument. IL-1β Level Determination. The cortex was homogenized in 0.9% NaCl, and the supernatant was collected after centrifuging for 10 min at 3000 rpm. IL-1β level was measured with ELISA kits (Westang Company, China). Data Analysis. One-way analysis of variance (ANOVA) followed by the Dunnett’s post-hoc test for comparing all groups to a control group was used to determine significant differences. The data are presented as the mean ± SE. To analyze interrelationships between physicochemical and molecule biological variables, bivariate relationships were investigated using Pearson’s correlation. For all statistical analyses, differences were considered significant when p < 0.05*, p < 0.01**, and p < 0.001***.

Article

RESULTS Induction of Ischemia-related Gene Expression and Neuronal Injuries by PM10. ET-1 mRNA levels increased after PM10 exposure in a season-dependent manner, and a significant difference was observed after winter sample treatment (p < 0.05, n = 6). In contrast, eNOS mRNA expression decreased after treatment and was attenuated to 0.55-, 0.77-, 0.50-, and 0.71-fold of the control by samples collected from spring, summer, autumn, and winter, respectively (Figure 1A). ET-1 protein expression presented the same

Figure 1. Effects of PM10 exposure on ET-1 and eNOS mRNA and protein expression in the rat brain. Male Wistar rats were instilled with a PM10 suspension from different seasons, and the final exposure concentration reached 10 mg/kg bw. The control group was instilled with the same amount of physiological saline, and the other special control group (vehicle group) was treated with the same amount of suspension from extracts of a “blank” filter. Instillation was performed five times for three days each. Each treatment had six samples, and each PCR or ELISA reaction was carried out in duplicate. The resulting values for each treated group were expressed as a fold increase compared to the mean values of the control group, which have been given an arbitrary value of 1. The control reported in the figure represents the normal control, and no significant difference was observed between the normal control and vehicle control group (p > 0.05, n = 6). The data are expressed as the means ± SE (n = 6); *p < 0.05 and **p < 0.01 vs the negative control.

trend with mRNA levels (Figure 1B), and the statistical differences were shown after autumn and winter sample treatment (for autumn, p < 0.05; for winter, p < 0.01, n = 6); eNOS protein levels reduced after PM10 exposure, and significant differences were observed after other sample treatment except for summer. As shown in Figure 2A, iNOS mRNA expression was significantly elevated after spring, autumn, and winter PM10 sample treatment, but the most obvious increase was observed after winter sample exposure (2.26-fold of the control, p < 0.001, n = 6). In addition, all PM10 samples tended to increase the COX-2 mRNA level in a season-dependent manner, but no significant difference was observed, even after winter sample treatment. Following this, we further tested the release of cytokine Il-1β, as well as the synthesis of adhesion molecule ICAM-1. Il-1β and ICAM-1 mRNA expression statistically increased after winter sample treatment (for Il-1β, 2.42-fold of the control, p < 0.001, n = 6; for ICAM-1, 1.94-fold of the control, p < 0.01, n = 6), but remained unchanged after other sample exposure. For protein expression (Figure 2B), iNOS, COX-2, Il-1β, and ICAM-1 protein levels increased after PM10 exposure in a season-dependent manner, and all of the peak values were observed after winter sample treatment (for iNOS, C

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 2. Effects of PM10 exposure on iNOS, COX-2, Il-1β, and ICAM-1 mRNA and protein expression in the rat brain. Male Wistar rats were instilled with a PM10 suspension from different seasons, and the final exposure concentration reached 10 mg/kg bw. The control group was instilled with same amount of physiological saline, and the other special control group (vehicle group) was treated with the same amount of suspension from extracts of the “blank” filter. Instillation was performed five times for three days each. Each treatment had six samples, and each PCR or ELISA reaction was carried out in duplicate. The resulting values for each treated group were expressed as a fold increase compared to the mean values of the control group, which have been given an arbitrary value of 1. The control reported in the figure represents the normal control, and no significant difference was observed between the normal control and vehicle control group (p > 0.05, n = 6). The data are expressed as the means ± SE (n = 6); *p < 0.05, **p < 0.01, and ***p < 0.001 vs the negative control.

increased in a season-dependent manner (20.45 ± 1.02%, 12.57 ± 0.9%, 23.06 ± 0.79%, and 26.41 ± 0.61% immunoreactive neurons for spring, summer, autumn, and winter, respectively). Impairment of Ischemia-Related Neuronal Function Caused by PM10 Exposure. Following the endothelial dysfunction and inflammatory response, we further detected the changes of neuronal function associated with cerebral ischemia. TEM observations (Figure 5) show that the synaptic structural parameters were changed after PM10 exposure in a season-dependent manner, and the most obvious difference was observed after winter sample treatment. Chemical Analysis of PM10 from Different Seasons. On the basis of the above season-dependent neuronal insults similar to that of cerebral ischemia, we further detected the chemical composition of PM10 samples during different seasons, including the contents of inorganic ion, element, PAHs, and carbon. As indicated in Table 2−5, the significant seasonal difference occurred in the levels of PAHs and carbon, and the winter sample showed the highest PAH load and carbon content. Furthermore, we conducted correlation analysis between the sensitive biomarkers and PM10 physicochemical characteristics (Table 6-9), and found that the maximum correlation coefficient occurred between ischemia-related injuries with PAHs and carbon levels. Considering that winter PM10 was mainly characterized by the highest PAHs and carbon contents, it implies that among different season samples, winter PM10 in a coal-burning city played the major role for contributing to ischemia-like injuries in the brain. Winter PM10 Sample-Aggravated Injuries on an Ischemic Neuron Model in Vitro. Because of the difficulty in establishing a MCAO rat model followed by intratracheal instillation of PM samples, we applied the OGD model by using primary cultured cortical neurons to stimulate the ischemic status to confirm the above winter PM10 caused

1.91-fold of the control, p < 0.01; for COX-2, 1.58-fold of the control, p < 0.05; for Il-1β, 2.61-fold of the control, p < 0.001; for ICAM-1, 1.59-fold of the control, p < 0.01, n = 6) To confirm the above results from gene analysis, we first used immunostaining images to provide further evidence (Figure 3).

Figure 3. Immunofluorescence staining in the cortex of rats from the vehicle control and PM10 sample treatment groups. The brain from different treatment groups was rapidly removed, washed several times with 0.01 M PBS (pH 7.4), fixed in 10% formalin for 24 h at room temperature, dehydrated by graded ethanol, and embedded in paraffin. Sections (5- or 6-μm-thick) were deparaffinized with xylene, stained with ET-1 antibody (red), DAPI (blue), and then observed by an Olympus confocal microscope. The images were captured using Image Pro Plus software with 400× magnification (Scale bar = 10 μm).

Images of immunostaining demonstrated that winter PM10 promoted the greatest expression of ET-1 protein (red fluorescent staining) than the other treatment groups, and this result was consistent with PCR and immunoblot analysis data. Following this, quantitative analysis of the morphological characteristic changes and consequent neuronal injuries were determined by TUNEL staining (Figure 4). Little specific staining occurred in the control (6.47 ± 0.32%), and the number of TUNEL-positive neurons (brunette nuclei) D

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 4. TUNEL staining in the cortex of rats from the vehicle control and PM10 sample treatment groups. Tissue sections of rat brains were fixed with 4% paraformaldehyde and embedded in paraffin, and then processed for the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay according to the manufacturer’s instructions. After developing with the diaminobenzidine (DAB) substrate, the slides were also counterstained with hematoxylin and observed by light microscopy with 400× magnification (Scale bar = 10 μm). The control reported in the figure represents the normal control, and no significant difference was observed between the normal control and vehicle control group (p > 0.05, n = 6). The data are expressed as the means ± SE (n = 6); *p < 0.05, **p < 0.01, and ***p < 0.001 vs the negative control.

Figure 5. Morphological characteristics of synapses in the cortex of rats from the vehicle control and PM10 sample treatment groups. About 1 mm × 1 mm × 1 mm pieces were rapidly cut from the cortex in the brain, fixed in 4% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 2 h at room temperature, and then postfixed in 2.5% osmium tetroxide in 0.1 M PB. After that, they were en bloc stained with 2% aqueous uranyl acetate for 1 h at room temperature in the dark, dehydrated by graded ethanol, and embedded in beam capsules. Sections, 70−80 nm-thick, were cut from the embedded tissue and collected onto grids to air-dry overnight. The grids were stained with uranyl acetate for 15−30 min and lead citrate for 3−15 min, and then the synaptic structural parameters were observed and measured by a transmission electron microscope and image analysis instrument scale bar = 500 nm). The control reported in the figure represents the normal control, and no significant difference was observed between the normal control and vehicle control group.

ischemic neuron model group, p < 0.01, n = 3). Then, cleaved caspase-3, an important marker for neuronal apoptosis, was examined in neurons. As expected, a statistically significant cleaved caspase-3 occurred in the OGD neurons, while winter PM10 treatment of model neurons significantly enhanced the effects (2.54-fold of ischemic neuron model group, p < 0.001, n = 3).

Table 2. Contents of Inorganic Ions in PM10 Samples from Different Seasons inorganic ions (μg/m3)

spring

summer

autumn

winter

Na+ NH4+ K+ Mg2+ Ca2+ F− Cl− SO42− N03−

2.471 4.290 2.456 10.868 99.078 0.364 9.125 114.201 33.181

4.037 5.466 3.953 9.904 74.215 0.208 3.292 126.752 27.965

5.124 5.918 4.741 11.186 71.168 0.612 12.146 121.143 23.958

1.476 6.226 4.238 13.651 93.296 0.483 46.377 56.625 17.424

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DISCUSSION Recent studies imply that air pollution, even in low concentration (below current Environmental Protection Agency (EPA) safety standards), could amplify the risk for ischemic stroke.31,32 However, while PM and O3 regulate the level of genes involved in key vasoregulatory pathways in the brain, the mechanisms of the pathology are still unknown.33 In addition, the particle components, attached by several toxic compounds, vary with the change of the source, the geographic location, and the season of PM sample collection.34 Therefore, seasonal variation in PM10 composition might affect the risk of inducing ischemic injuries in the brain. To test this hypothesis, we treated Wistar rats with PM10 samples collected during different seasons in Taiyuan, a typically coal-burning city of North China, and first analyzed the ischemic markers in the rat

effects. Compared with the cortical ischemic neuron model group (Figure 6), iNOS protein expression was statistically elevated by winter PM10 sample treatment (2.46-fold of the ischemic neuron model group, p < 0.01, n = 3). COX-2 and ICAM-1 expression was statistically amplified, and the enhancement varied by the season in which the PM10 sample was obtained. The winter PM10 sample showed the most obvious tendency (1.68-fold of ischemic neuron model group for COX-2, p < 0.001, n = 3, and 1.43-fold for ICAM-1 in the E

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology Table 3. Element Contents in PM10 Samples from Different Seasons

Table 4. Contents of PAHs in PM10 Samples from Different Seasons

elements (μg/m3)

spring

summer

autumn

winter

PAHs (ng/m3)

spring

summer

autumn

winter

Zr Al Sr Mg Ti Ca Fe Ba Si Li Be Na P K Sc V Cr Mn Co Ni Cu Zn As Rb Y Mo Cd Sn Sb Cs La Ce Sm W Tl Pb Bi Th U

1.871 179.115 2.277 65.643 9.498 147.236 16.359 5.212 413.110 0.246 0.003 8.060 0.747 8.134 0.029 0.105 0.030 0.465 0.008 0.027 0.070 0.238 3.143 0.033 0.068 0.089 0.005 0.025 0.058 0.006 0.360 0.477 0.016 0.008 0.001 0.391 0.013 0.034 0.017

1.368 104.166 1.413 42.229 6.282 112.887 8.033 3.319 325.649 0.214 0.004 26.839 0.632 10.735 0.028 0.082 0.041 0.313 0.005 0.019 0.061 3.808 2.550 0.023 0.048 0.063 0.005 0.018 0.052 0.005 0.247 0.347 0.011 0.016 0.001 0.295 0.009 0.025 0.012

0.616 59.808 0.882 24.514 2.803 86.904 6.424 2.216 302.157 0.220 0.008 86.236 0.743 24.677 0.023 0.057 0.035 0.332 0.004 0.020 0.100 15.084 1.624 0.028 0.017 0.011 0.010 0.016 0.062 0.006 0.053 0.163 0.005 0.042 0.002 0.333 0.006 0.017 0.006

0.651 155.185 2.423 56.791 4.115 127.442 12.510 2.660 359.138 0.475 0.002 5.753 1.174 8.053 0.018 0.066 0.042 0.361 0.006 0.023 0.163 0.412 2.558 0.029 0.022 0.006 0.005 0.094 0.190 0.007 0.070 0.115 0.006 0.012 0.002 0.473 0.030 0.021 0.007

acenaphthylene (ANY) fluorene (FLU) benzo(g,h,i)perylene (BPE) indeno(1,2,3-cd)pyrene (IPY) dibenzo(a,h)anthracene (DBA) benzo(b)fluoranthene (BbF) coronene (COR) phenanthrene (PHE) anthracene (ANT) fluoranthene (FLT) benzo(a)anthracene (BaA) chrysene (CHR) pyrene (PYR) benzo(a)pyrene (BaP) benzo(e)pyrene (BeP) benzo(k)fluoranthene (BKF)

0.216 0.413 12.353 8.919 2.983 20.458 6.630 2.643 0.300 9.723 9.441 11.879 7.821 7.724 8.449 3.545

0.104 0.246 4.658 3.636 1.328 9.575 2.288 1.206 0.095 2.781 1.867 3.979 1.962 2.277 3.687 1.429

0.327 0.640 24.071 22.019 9.491 51.826 25.166 6.974 1.257 25.298 23.426 41.831 26.138 18.286 19.259 9.269

0.810 1.086 53.398 39.421 18.006 76.266 30.197 28.438 7.473 77.157 66.413 64.240 86.676 41.063 31.143 13.141

Table 5. Carbon Content in PM10 Samples from Different Seasons carbon (μg/m3)

spring

summer

autumn

winter

OC EC TC

39.434 56.926 17.491

16.034 25.495 9.461

44.162 65.185 21.024

77.153 111.333 34.180

Table 6. Pearson’s Correlation between Inorganic Ion Level and Biological Markers +

Na NH4+ K+ Mg2+ Ca2+ F− Cl− SO42− NO3−

ET-1

iNOS

IL-1β

ICAM-1

COX-2

−0.584 0.364 0.141 0.939 0.527 0.713 0.873 −0.841 −0.634

−0.729 0.271 0.002 0.944 0.662 0.568 0.895 −0.892 −0.577

−0.592 0.492 0.250 0.981* 0.459 0.675 0.937 −0.901 −0.747

−0.815 0.356 0.040 0.972* 0.651 0.407 0.961* −0.974* −0.660

−0.045 0.881 0.791 0.771 −0.189 0.790 0.744 −0.630 −0.929

brain after ischemic stroke,37 whereas it is reported that focal cerebral ischemia creates an area of infarction that is surrounded by neuronal tissue that may respond to nearby damage by creating new synapses and lead to partial recovery of neuronal function.38 Physiologically, cortical connections after ischemia become hyperexcitable. Anatomically, ischemic lesions induce axonal sprouting within local, intracortical projections and long distance, interhemispheric projections. This postischemic axonal sprouting establishes substantially new patterns of cortical connections with deafferented or partially damaged brain areas. Axonal sprouting after ischemic lesions is induced by a transient pattern of synchronous, low-frequency neuronal activity in a network of cortical areas connected to the infarct.39 Therefore, we further determined the number of TUNEL-positive neuron and morphological alterations. Consistent with the changes of endothelial and inflammatory genes, all samples tended to cause neuronal apoptosis accompanied by stimulating synaptic function, but the most obvious statistical difference was observed after winter sample inhalation. The above-mentioned ischemia-related effects resulting from PM10 changed as a function of season. The chemical

cortex, including endothelial function, inflammatory response, and subsequent neuronal function. ET-1 and eNOS are important endothelial markers for the constriction of cerebral blood vessels and the development of cerebral infarction.35 A marked inflammatory reaction, including proinflammatory enzyme activation, cytokine release, and adhesion molecule synthesis, promoted blood-borne inflammatory cell adherence and infiltration, and exacerbated brain injury by physically obstructing capillaries and reducing blood flow during reperfusion and/or by migrating into the brain parenchyma and releasing cytotoxic products.36 In the present study, imbalanced ET-1 and eNOS expression and exacerbated iNOS, Il-1β, and ICAM-1 releases suggest the reduction of cerebral blood and a tendency to ischemic injuries via endothelial dysfunction and inflammatory response. Interestingly, the actions showed season-dependent properties, and the winter PM10 sample caused the most obvious changes. Ischemic injuries result in cell death and disability, and apoptosis is an important contributor to neuronal death in the F

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology Table 7. Pearson’s Correlation between Elements Level and Biological Markers Zr Al Sr Mg Ti Ca Fe Ba Si Li Be Na P K Sc V Cr Mn Co Ni Cu Zn As Rb Y Mo Cd Sn Sb Cs La Ce Sm W Tl Pb Bi Th U

ET-1

iNOS

IL-1β

ICAM-1

COX-2

−0.505 0.338 0.507 0.275 −0.332 0.182 0.373 −0.224 0.252 0.808 −0.218 −0.154 0.893 −0.060 −0.832 −0.342 0.031 0.286 0.211 0.452 0.888 −0.123 −0.053 0.657 −0.472 −0.588 0.057 0.783 0.806 0.991** −0.514 −0.577 −0.402 −0.019 0.909 0.909 0.747 −0.250 −0.380

−0.376 0.504 0.662 0.447 −0.185 0.355 0.515 −0.090 0.398 0.864 −0.399 −0.340 0.923 −0.250 −0.780 −0.197 0.038 0.378 0.365 0.557 0.867 −0.311 0.131 0.689 −0.334 −0.469 −0.134 0.843 0.843 0.996** −0.381 −0.469 −0.260 −0.210 0.875 0.971* 0.833 −0.098 −0.241

−0.594 0.291 0.492 0.232 −0.422 0.126 0.288 −0.333 0.159 0.877 −0.246 −0.168 0.945 −0.080 −0.906 −0.433 0.198 0.149 0.125 0.338 0.952* −0.134 −0.096 0.529 −0.556 −0.673 0.026 0.856 0.885 0.986* −0.598 −0.672 −0.491 −0.021 0.964* 0.908 0.809 −0.340 −0.474

−0.386 0.544 0.730 0.498 −0.191 0.393 0.492 −0.131 0.371 0.960* −0.544 −0.474 0.981* −0.396 −0.813 −0.203 0.246 0.263 0.354 0.474 0.906 −0.443 0.200 0.535 −0.335 −0.478 −0.297 0.948 0.940 0.966* −0.382 −0.498 −0.266 −0.337 0.893 0.984* 0.943 −0.103 −0.254

−0.963* −0.346 −0.106 −0.397 −0.893 −0.501 −0.367 −0.838 −0.486 0.619 0.257 0.356 0.702 0.415 −0.944 −0.898 0.484 −0.442 −0.516 −0.295 0.872 0.392 −0.660 0.009 −0.951* −0.985* 0.470 0.604 0.698 0.673 −0.966* −0.978* −0.925 0.500 0.895 0.470 0.473 −0.850 −0.916

Table 8. Pearson’s Correlation between PAH Level and Ischemia-Related Markers acenaphthylene (ANY) fluorene (FLU) benzo(g,h,i)perylene (BPE) indeno(1,2,3-cd)pyrene (IPY) dibenzo(a,h)anthracene (DBA) benzo(b)fluoranthene (BbF) coronene (COR) phenanthrene (PHE) anthracene (ANT) fluoranthene (FLT) benzo(a)anthracene (BaA) chrysene (CHR) pyrene (PYR) benzo(a)pyrene (BaP) benzo(e)pyrene (BeP) benzo(k)fluoranthene (BKF)

ET-1

iNOS

IL-1β

ICAM-1

COX-2

0.905 0.934 0.916

0.906 0.905 0.898

0.963* 0.979* 0.970*

0.950* 0.915 0.927

0.805 0.871 0.853

0.912

0.870

0.963*

0.878

0.909

0.893

0.851

0.952*

0.870

0.915

0.904

0.839

0.947

0.823

0.942

0.855 0.848 0.827 0.880 0.894

0.757 0.860 0.846 0.877 0.886

0.889 0.923 0.905 0.947 0.956*

0.711 0.934 0.931 0.931 0.932

0.970* 0.784 0.759 0.825 0.833

0.892 0.868 0.910 0.918 0.905

0.825 0.866 0.888 0.867 0.834

0.939 0.939 0.966* 0.963* 0.943

0.814 0.926 0.916 0.861 0.809

0.950 0.824 0.867 0.920 0.945

Table 9. Pearson’s Correlation between Carbon Level and Biological Markers OC EC TC

ET-1

iNOS

IL-1β

ICAM-1

COX-2

0.983* 0.972* 0.980*

0.975* 0.956* 0.970*

0.999** 0.998** 0.999**

0.961* 0.949 0.958*

0.764 0.806 0.776

If the implication is reasonable, there are correlations between winter PM10 and the development and progression of cerebral ischemia, then the winter sample might show the most serious aggravation of the injuries from the ischemic model. To test this hypothesis, we established ischemic neuron models and treated the models with PM10 collected during different seasons, and the related gene expression and neuronal injuries were investigated. Interestingly, based on the successful OGD neuron model, only the winter PM10 sample significantly increased the tendency of COX-2, iNOS, and ICAM-1 expression. Previous studies have demonstrated that the expressions of caspase genes are higher in both early and late stages of ischemia, and ischemic injury could be restrained by genetic manipulations or drugs via blocking caspase family members.40 As the members of the identified caspase family, caspase-1 and caspase-3 seem to play a critical role in ischemiamediated apoptosis. Therefore, we investigated the sequent injury outcome by detecting the expression of cleaved caspase3. OGD model neurons showed a cleavage of caspase-3, while winter PM10 exposure significantly amplified the effects. In summary, the results indicate that PM10 samples caused season-dependent endothelial dysfunction and inflammatory response, following neuronal functional alteration in the rat cortex. Winter PM10, which was characterized by higher levels of PAHs and carbon, showed a significant correlation with these changes and played the most important role in aggravating ischemia-like brain injuries. These results provide experimental evidence for clarifying the association between season-dependent PM10 pollution and increased risk of ischemia-like injuries

components of PM10 collected in the same location were seasonally specific. Therefore, the various behaviors of PM10 samples may be due to their different physicochemical characteristics. To clarify the issue, we analyzed the chemical composition of PM10 samples from different seasons and found that the significant difference among different seasons existed in PAHs and carbon contents, with the winter sample carrying the highest PAHs and carbon loading. To provide further evidence for the explanation, we conducted a correlative analysis between the above ischemia-related biomarkers and PM10 physicochemical characteristics and observed the maximum correlation coefficient between ischemia-related bioindices with PAHs and carbon levels. It implies that the changes in chemical properties of PM10 from different seasons contributed to the variations in their ischemia-like injuries, and higher levels of PAHs and carbon might be the major reason for winter PM10 inducing the more serious damages than samples from other seasons. G

DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

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Figure 6. Effects of PM10 exposure on the expression of iNOS, COX2, ICAM-1, and cleaved caspase-3 in OGD model neurons. Primary cultured cortical neurons were treated by oxygen and glucose deprivation (OGD) for 45 min and were reperfused for 24 h. Then, the model cells were divided randomly into five equal groups: four treatment groups were individually treated with 100 μg/mL PM10 from different seasons for 24 h, and the vehicle control group was treated with the same amount of 0.9% physiological saline. Every group was divided into three subgroups. The resulting values for each treated group were expressed as a fold increase compared to the mean values of the control group, which have been given an arbitrary value of 1. The control reported in the figure represents the normal control, and no significant difference was observed between the normal control and vehicle control group (p > 0.05, n = 6). The data are expressed as the means ± SE (n = 6); *p < 0.05, **p < 0.01, and ***p < 0.001 vs negative control.

and substantiates the notion that winter PM10 exposure made for the development and deterioration of cerebral ischemia.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-351-7011932. Fax: +86-351-7011932. E-mail: [email protected]. Funding

This study was supported by National Natural Science Foundation of China (Nos. 21477070, 21377076, 21307079, and 21222701), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, Nos. 20121401110003 and 20131401110005), and Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 20120201). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS PM10, particulate matter; PAHs, polycyclic aromatic hydrocarbons; ET-1, endothelin-1; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; Il-1β, interleukin-1β; ICAM-1, intercellular adhesion molecule 1; OGD, oxygen and glucose deprivation; DMEM, Dulbecco’s modified Eagle’s medium H

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DOI: 10.1021/tx500392n Chem. Res. Toxicol. XXXX, XXX, XXX−XXX