676

Introduction

The link between elevated concentrations of air pollution and adverse public health effects is now well established. Increased levels of particulate matter (PM10 and PM2.5) are widely recognized for their causal contribution to mortality and cardiovascular morbidity and mortality (Pope et al., 2009; Brook et al., 2010). Important legislative efforts have been made in most industrialized countries to reduce emissions of pollutants and to determine safe

levels. Although it remains unclear which pollutants or fraction of particles are driving the health effects that are identified in epidemiological studies, strong circumstan-tial evidence indicates that traffic plays an important role.

In addition, evidence is accumulating that exposure to air pollution can cause brain damage and neurodegeneration. Long-term exposure to air pollution is associated with neuroinflammation and a disruption of the blood-brain barrier (Calderón-Garcidueñas

RESEARCH ARTICLE

Changed gene expression in brains of mice exposed to traffic in a highway tunnel

Inge Bos1,2, Patrick De Boever1,3, Jan Emmerechts4,5, Jurgen Buekers1, Jeroen Vanoirbeek5, Romain Meeusen2, Martine Van Poppel1, Benoit Nemery5, Tim Nawrot3,5, and Luc Int Panis1,6

1Flemish Institute for Technological Research (VITO), Mol, Belgium, 2Department of Human Physiology & Sports Medicine, Vrije Universiteit Brussel, Brussels, Belgium, 3Centre for Environmental studies (CMK), Hasselt University, Diepenbeek, Belgium, 4Center for Molecular and Vascular Biology, University of Leuven, Belgium, 5Department of Public Health, Occupational and Environmental Medicine, Unit of Lung Toxicology, University of Leuven, Leuven, Belgium, and 6Transportation Research Institute (IMOB), Hasselt University, Diepenbeek, Belgium

AbstractContext: Air pollution has been suggested to have an impact on the brain.

Objective: The objective was to assess the expression of inflammation-related genes in the brains of mice that had been exposed for 5 days to a well-characterized traffic-polluted environment, i.e. a highway tunnel.

Materials and methods: Twenty C57BL6 mice were randomly allocated to four groups of five animals. Two groups were placed in the tunnel for 5 days (mean PM 2.5, 55.1 μg/m3, mean elemental carbon, EC 13.9 μg/m3) in cages with or without filter, two control groups were housed outside the tunnel. Animals were assessed within 24 hours after the last exposure day. Lung injury and inflammation were assessed by bronchoalveolar lavage (BAL) and histology. Blood leukocytosis and coagulation parameters were determined in peripheral blood. The olfactory bulb and hippocampus were analyzed for changes in expression of inflammatory genes and brain-derived neurotrophic factor (BDNF).

Results and discussion: Although carbon particles were abundant in alveolar macrophages of exposed mice and absent in non-exposed mice, there was no evidence of pulmonary or systemic inflammation. There was an increased expression of genes involved in inflammatory response (COX2, NOS2, NOS3, and NFE2L2) in the hippocampus of the exposed mice. In the olfactory bulb, a downregulation was found for IL1α, COX2, NFE2L2, IL6, and BDNF.

Conclusion: Although this short-term exposure to traffic-related pollution did not induce pulmonary or systemic inflammation, the expression of inflammatory genes was affected in different brain areas. The decreased BDNF expression in the olfactory bulb suggests lower brain neurotrophic support in response to traffic-related air pollution.

Keywords: Particulate matter, black carbon, brain-derived neurotrophic factor (BDNF), inflammation, hippocampus, olfactory bulb, blood, bronchoalveolar lavage (BAL), lung

Address for Correspondence: Luc Int Panis, VITO, Environment and Health, Boeretang 200, 2400 Mol, Belgium. Tel: +32 14 335102. Fax: +32 14 58 26 57. E-mail: luc.intpanis@vito.be

(Received 31 May 2012; revised 11 July 2012; accepted 17 July 2012)

Inhalation Toxicology, 2012; 24(10): 676–686© 2012 Informa Healthcare USA, Inc.ISSN 0895-8378 print/ISSN 1091-7691 onlineDOI: 10.3109/08958378.2012.714004

Inhalation Toxicology

24

10

676

686

31May2012

11July2012

17July2012

0895-8378

1091-7691

© 2012 Informa Healthcare USA, Inc.

10.3109/08958378.2012.714004

2012

Changed gene expression in mice brains

I. Bos et al.

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

Changed gene expression in mice brains 677

© 2012 Informa Healthcare USA, Inc.

et al., 2004, 2007, 2008a). Calderón-Garcidueñas and colleagues were among the first to identify cognitive deficits in children exposed to ambient air pollution in Mexico City and they also evidenced prefrontal lesions using MRI analysis (Calderón-Garcidueñas et al., 2008b, 2011). Other research groups confirmed associations between long-term exposure to air pollution and cognitive impairments in healthy adults, children and older women (Suglia et al., 2008; Chen & Schwartz, 2009; Ranft et al., 2009; Weuve et al., 2012). Oxidative stress-mediated inflammation is thought to contribute to the mode of action (Campbell et al., 2005; Mohankumar et al., 2008; Block & Calderón-Garcidueñas, 2009; Brook et al., 2010). However, the exact pathways of toxicity that lead to neurological effects following exposure to traffic-related air pollution remain largely unknown.

Animal experiments are instrumental to confirm epi-demiological associations and to elucidate the sequence of events that link exposure to physiological changes, effects on the brain, and ultimately health effects.

In this study, we exposed mice to unaltered traffic-related air pollution in a highway tunnel. Experiments in tunnels have the advantage that boundary conditions such as air flow and traffic volume are well known. The air pollution in tunnels is representative for overall vehi-cle fleet emissions (Phuleria et al., 2006). Furthermore, the suitability of road tunnels for determining real world emissions for road vehicles has been demonstrated in various studies (e.g. Gertler et al., 2001; Sturm et al., 2001), but only a few have measured PM (Weingartner et al., 1997). Tunnel studies eliminate the variability due to meteorological effects that may complicate the use of ambient air monitoring measurements for determining concentrations near busy roads. As a result, road tunnels form an ideal case to test the effects of traffic exposure on physiological endpoints in laboratory animals.

We hypothesized that mice exposed to traffic-related air pollution would show an inflammatory response and a pro-inflammatory gene response in the brain.

Methods

Test siteThe experiment was conducted in the Craeybeckx tunnel in Antwerp (Belgium) between 23rd and 28th June 2010. The Craeybeckx tunnel links the major ring road around Antwerp (R0) with the southbound A1/E19 (Antwerp-Brussels). This tunnel, built in 1981, is about 1600 meters long with a slightly curved trajectory and is divided in two separate tubes, one for each direction, with four driving lanes per tube. The speed limit inside the tunnel is 100 km.h−1. Our experiment was conducted in the east bore of the tunnel (Figure 1), which carries the highway traffic northbound towards Antwerp. On a typical day during the experiment, the average traffic stream consisted of 2971 vehicles/hour that varied from 260 vehicles/hour at night, up to 6190 vehicles/hour during morning rush hour. We measured an average of 2270 vehicles/hour

(max 5200 vehicles/hour) of passenger cars, 272 vehicles/hour (max 637 vehicles/hour) of light duty vehicles, and 430 vehicles/hour (max 812 vehicles/hour) of heavy duty vehicles. During the experiment there was only natural ventilation in the tunnel, the automated ventilation sys-tem was not in operation during the sampling period.

Air quality measurementsPM2.5 mass concentration measurements were performed using a low volume filter sampler (Partisol, Thermo). Filters (type Tissu Quartz) were subsequently analyzed for elemental carbon (EC) and organic carbon (OC) by TOT (Thermo Optical Transmission, Sunset) using the EPA/NIOSH protocol (Peterson & Richards, 2002).

Total particle number and size resolved ultrafine particle (UFP) concentration measurements were per-formed using a Scanning Mobility Particle Sizer (TSI SMPS; Model 3936) comprising DMA 3081 and CPC 3786. This configuration measures a size range of 9.82 nm to 414.2 nm. In addition to the stationary measure-ments, short-time measurements using a hand-held CPC (P-Trak, TSI, 20 nm–1000 nm) were performed in the cages with and without filter, and in the tunnel air in order to assess mice exposure. The concentrations in the tunnel were compared to those measured in the cage with the reference mice group B at the Traffic Control Centre in Antwerp.

Experimental set-upA 5-day exposure study was performed with 20 male, 6 week-old C57BL6 mice. All animals were weighed and identified before exposure. The animals were randomly allocated to four groups:

• Test group: five mice in a polyethylene cage without filter cap were placed in the tunnel.

• Control group (A): five mice in a cage with a “rein-forced” (2 × 3 layers) filter cap were also placed in the

Figure 1. Experimental set-up and animal cages. Second cage from the left is the control group with reinforced filter cap (control group A). Third cage from the left is the test group, left and right cages are empty. (source: Flemish Traffic Control Centre).

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

678 I. Bos et al.

Inhalation Toxicology

tunnel. The TECNIPLAST patented Filter Top (Mini-IsolatorTM) Cage system has a atmospheric dust effi-ciency of 92% for retention of 8–10 μm particles.

• Control group (B): five mice in a cage with normal fil-ter cap were placed inside the building of the Traffic Control Centre in Antwerp near the tunnel.

• Control animal facility: five animals that stayed in the animal house in Leuven during the experiment were used as a third control group.

Animals from the groups A, B, and test group were transported by car from Leuven to Antwerp (approxi-mately 1 hour drive). The test group and control group A were placed inside the tunnel in one of the small areas, situated at every 100 meters inside the tunnel, giving access to emergency escapes (Figure 1). Animals received food and water ad libitum and were housed on a bedding consisting of wood chips.

This set-up using two additional controls (one in the tunnel but shielded from particulate air pollution to control for differences in stress experienced from being in the tunnel (control A), and another placed in a build-ing near the tunnel (control B)) allows to discriminate between the effect of being inside the tunnel (including exposure to noise and stress) and the effects of exposure to fresh traffic-related air pollution.

At the end of the exposure period, all animal groups were transported back to the laboratory in Leuven, where autopsies were carried out immediately upon arrival. The mice were first weighed and then blood samples were obtained from the retro-orbital sinus under anes-thesia (sodium pentobarbital, 60 mg/kg, i.p.). Blood cell counts and differentials were determined using a Cell-Dyn 3500R counter (Abott, Diegem, Belgium). The activated partial thromboplastin time (aPTT) and the prothrombin time (PT) assays were performed on the BCS-XP coagulation analyzer (Siemens, Hamburg, Germany) using the manufacturer’s reagents (Actin FS and Innovin, respectively) and adapted procedures for murine coagulation testing. Fibrinogen concentrations were also determined on the BCS-XP by the functional Clauss method, calibrated with Standard Human Plasma (Siemens, Hamburg, Germany). Next, bronchoalveo-lar lavage (BAL) was performed to assess pulmonary inflammation. The left bronchus was ligated and the tra-chea exposed and cannulated with a 20 Gauge cannula to lavage the right lung three times with 0.4 mL of sterile NaCl 0.9%. The cells in fresh BALF were stained with trypan blue and counted in a Burker hemocytometer. Cell differentials were determined by light microscopy on cytocentrifuge preparations fixed in methanol and stained with Diff Quick (Siemens, Brussels, Belgium). Airway macrophages were visualized by light microscopy (AxioPlan 2 Imaging, Zeiss, Zaventem, Belgium) and pic-tures were taken with Axiovision Rel. 4.6. (Zeiss).

After the BAL, the left lung was instilled, in situ, with 0.4 mL paraformaldehyde 4% (PFA) until full expansion as assessed visually, and then removed and immersed

in PFA 4% for fixation. Subsequently, the lung tissue was embedded in paraffin and 8 μm cross-sections were stained with hematoxylin-eosin for histological analy-sis. The sections were analyzed with a Zeiss AxioPlan 2 Imaging microscope (Zeiss, Jena, Germany). The degree of inflammation of the lung tissue was estimated based on typical characteristics of inflammation. For the inves-tigations of the lung tissues by electron microscopy, small biopsies of the alveolar region were also taken.

Immediately after removal of the lungs, the brains of the animals from control group A, B, and the test group were removed from the skull. The hippocampus and the bulbus olfactorius were dissected for gene expression analysis. Following dissection, the hippocampus and the bulbus olfactorius were stored overnight at 4°C in RNA later to stabilize the RNA (Life Technologies, Ghent, Belgium). Next, the RNA later was removed by decanta-tion and samples were stored immediately at −80°C.

Protocols were in accordance with national rules on animal experiments and were approved by the Ethics Committee on Animal Experiments of the Faculty of Medicine the University of Leuven (Belgium).

Gene expression analysis with real-time PCRRNA was extracted from the brain samples by Ambion® RiboPure™ kit (Life Technologies, Ghent, Belgium) according to the manufacturer’s instructions. Briefly, tis-sue samples were weighed and homogenized in 10–20 volumes of TRI reagent using a rotor-stator homog-enizer. Total RNA was extracted from the homogenate with glass-fiber filter purification methodology and eluted with a low salt buffer. The RNA concentration was determined using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, USA). The qual-ity and integrity of the extracted RNA was tested with capillary gel electrophoresis using RNA 6000 Nano Chips (Agilent Technologies, Palo Alto, CA, USA), analyzed on the Agilent 2100 BioAnalyzer. RNA was stored in elution solution (Life Technologies) at −80°C until further use. The synthesis of cDNA started from 1 μg total RNA using a combination of oligo (dT) 18 primers and random hex-amers. This was done with the Transcriptor First Strand cDNA Synthesis Kit and following the instructions of the manufacturer (Roche Applied Sciences, Vilvoorde, Belgium). All reverse transcription reactions were done in a Veriti 96-well Thermal Cycler of Life Technologies. Real-time PCR amplifications were in 20 µL aliquots in 96-well plates using LightCycler 480 (Roche Applied Sciences). The reaction mixtures consisted of 5 µL cDNA sample and 15 µL reaction mix (10 µL Lightcycler 480 Probes Master Kit (Roche Applied Sciences)), 1 µL primer-probe mix, and 5 µL nuclease-free water). We have used PrimeTime™ assays from Integrated DNA Technologies (Leuven, Belgium). The real-time PCR pro-tocol consisted of one cycle of preincubation (10 min at 95°C), 45 cycles of amplification (10 s at 95°C, 30 s at 62°C, and 1 s at 72°C), and one final cycle (10 s at 40°C). All analyses were run in triplicate. Real-time PCR analysis

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

Changed gene expression in mice brains 679

© 2012 Informa Healthcare USA, Inc.

generated sample specific crossing points (Cp). In the LightCycler software the “Second Derivative Maximum Method” is performed where Cp is automatically identi-fied and measured at the maximum acceleration of fluo-rescence (Rasmussen, 2001).

Details of genes that were analyzed, and the 5’ exo-nuclease PrimeTime™ assays that were used are given in Table 1 and Supplementary Table S1. PCR efficiency was considered 100% for the 5’-exonuclease assays. Three technical replicates of the real-time PCR analysis were averaged. Stability of the putative housekeeping genes (B2M, GAPDH, HPRT1, YWHAZ) was verified using geNorm (Vandesompele et al., 2002). The normalized, relative gene expression ratio (R) was calculated using the Relative Expression Software Tool (REST© 2009 V.2.0.13) (Pfaffl et al., 2002). REST calculates the normalized, rela-tive gene expression ratio between any two groups using the mathematical model:

Concentration efficiencygeneaverage C group average C grp p= −( ) (1 ooup2)

Relative expression ratio target gene

arg

=Concentration t et geene

Geomeanof concentrationof housekeeping genes

GeomeanConcentration housekeeping gene

Concentrationhous=

1, ,…eekeeping gene n

n

with n the number of housekeeping genes that are con-sidered stabile in expression using geNorm. REST was used because of two advantages over other relative quan-tification strategies for RT-PCR data. REST implements a mathematical model that allows for relative quanti-fication between groups in contrast to previous math-ematical models that calculate relative expression ratios between two samples. Moreover, REST has implemented a valid statistical test for relative gene expression analysis using the Pair Wise Fixed Reallocation Randomization Test© (REST 2009 software user guide). Statistical tests to

Table 1. List of parameters selected for analysis in blood or bronchoalveolar lavage (BAL) and genes selected for the mRNA analysis in the olfactory bulb and hippocampus.

Gene/Code Name Tissue Analysis assaya/unit Expected change Reference

IL1α Interleukin 1α Brain mRNA + Campbell et al., 2005

BDNFb Brain-derived neurotrophic factor Brain mRNA − Bos et al., 2011

IL1β Interleukin 1β Brain mRNA + Calderón-Garcidueñas et al., 2008a

NFE2L2 Nuclear factor (erythroid-derived 2)-like 2

Brain mRNA + Ishii et al., 2000

COX2 Cyclooxygenase 2 Brain mRNA + Calderón-Garcidueñas et al., 2008a

TNF Tumor necrosis factor Brain mRNA + Campbell et al., 2005IL6 Interleukin 6 Brain mRNA +NOS2 Inducible nitric oxide synthase Brain mRNA + Mohankumar et al., 2008; van

Berlo et al., 2010NOS3 Endothelial nitric oxide synthase Brain mRNA

Neutrophil leukocytes BAL Count +Eosinophil leukocytes BAL CountBasophil leukocytes BAL CountLymphocytes BAL CountMonocytes BAL CountRed blood cells BAL PresenceMacrophages BAL Black carbon + Jacobs et al., 2010aNeutrophil leukocytes Blood Count + Jacobs et al., 2010bEosinophil leukocytes Blood CountBasophil leukocytes Blood CountLymphocytes Blood CountMonocytes Blood CountRed blood cells Blood CountHemoglobin Blood µg/dL

aPTT Coagulation Blood SecPT Coagulation Blood SecFBg Coagulation Blood mg/dLa PrimeTime™ assay used for quantifying gene expression. Assay name and reference sequence numbers are given in Supplementary Table S1. B2M, GAPDH, HPRT1, and YWHAZ were considered as housekeeping genes.

b In mice, the expression of the BDNF gene is controlled by different promoters (depending on tissue type and activity) resulting in the tran-scription of four different mRNA’s (Aid et al., 2007).

BDNF, brain-derived neurotrophic factor transcript 4; COX2, cyclooxygenase 2; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; NOS2, inducible nitric oxide synthase; NOS3, endothelial nitric oxide synthase; TNF, tumor necrosis factor. 

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

680 I. Bos et al.

Inhalation Toxicology

determine accuracy of relative expressions are complex because ratio distributions do not have a standard devia-tion. REST 2009 software overcomes this problem by using a statistical randomization test that has the advan-tage of making no assumptions about the distribution of the data, while remaining as powerful as parametric tests (Pfaffl et al., 2002). The software allows comparing two groups, with multiple data points (C

p) in the two groups,

and it also allows the calculation of the relative expres-sion ratio between them. For more detailed information on the statistical test, the reader is referred to the paper of (Pfaffl et al., 2002).

Statistical analysisStatistical analysis for cell counts in BALF, blood formula, and coagulation parameters was performed using non-parametric Kruskal–Wallis tests with Dunn’s multiple comparison post-tests. Statistical significance of the rela-tive gene expression ratio is analyzed using the Pair Wise Fixed Reallocation Randomization Test©, with 10,000 per-mutations (Pfaffl et al., 2002). Confidence intervals at 95% are obtained using bootstrapping techniques on the per-mutated expression data (REST 2009 software user guide).

Results

ExposureFigure 2A shows the daily concentrations of EC, OC, and total PM2.5 in the tunnel during and after the experi-ment. The daily PM2.5 concentration ranged from 39.4 to 66.7 μg/m3, with an average concentration of 55.1 μg/m3. The concentration of PM2.5 for control group B at the Traffic Control Centre is assumed to be similar to

the urban Air Quality monitoring station in the city of Antwerp (29.4 μg/m3), which is situated approximately 3000 m to the North.

The daily average EC concentration ranged from 9.5 µg/m3 (weekend) up to 18.3 µg/m3 (weekday). The aver-age concentrations for EC and OC were 23.1 μg/m3 and 13.9 μg/m3, respectively. The EC fraction ranged from 19 to 34%, and the OC accounts for 31–48%. Especially during weekdays, EC concentrations in the tunnel were much higher than average urban background values (2.04 µg/m3) reported in Flanders (Vercauteren et al., 2011). The total carbonaceous PM2.5 fraction in the tun-nel was 50–84% of the PM2.5 mass concentration. In peak hours, the concentrations of BC reached values of up to 150 μg/m3. In the night hours (2–3 AM), the BC values were around 14 μg/m3. UFP concentrations (Figure 2B) showed a good correlation with daily traffic intensities (peak hours vs. non-peak hours) and a clear daily and weekly variation (weekend versus weekdays).

Because of practical reasons, the UFP concentration was mainly measured in the middle of the tunnel and not in the cages. Additional measurements in the cage were performed during a short time using hand-held CPCs (Condensation Particle Counter) that measure number concentrations in the size range 20 nm–1000 nm. The results showed no differences between concentrations in the cage ((129 ± 16) 103 particles/cm3) and the tun-nel environment ((150 ± 25) 103 particles/cm3). The UFP concentrations measured in the tunnel (in and next to the cages) were 20 times higher than the concentrations measured at the Traffic Control Centre in Antwerp (6.103 particles/cm3).

BALF and blood analysisThe presence of carbon particles was clearly observed in the alveolar macrophages obtained by bronchoal-veolar lavage of the test group (Figure 3, Test group). No black carbon was visible in alveolar macrophages of control group A (Figure 3, control A) and control group B (Figure 3, control B), indicating that the reinforced cage protected the animals from being exposed to black carbon.

There were no significant differences in BAL leukocyte counts between animals of the test group, the control A, B, or animal facility. Also, leukocyte counts in blood did not differ between the test group and control B, and animal facility. Finally, coagulation parameters (aPTT, PT, and fibrinogen plasma levels) were not affected by PM exposure. A complete list of results for all parameters is presented in Supplementary tables S2, S3, and S4 for BAL, blood and coagulation parameters, respectively.

Lung histologyThe gross appearance of the lungs was normal in all animals and there were no histological differences between the various animal groups. Samples were also analyzed by electron microscopy. No PM was found in the alveolar region of the investigated alveoli. No

Figure 2. Daily elemental carbon (EC) and organic carbon (OC) fraction in PM2.5 (A), particle number concentration (PNC) in #/cm3 and vehicle numbers (vehicle/5 min) (B) measured in the tunnel.

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

Changed gene expression in mice brains 681

© 2012 Informa Healthcare USA, Inc.

signs of inflammation or increased presence of alveolar macrophages were observed, confirming histological observations.

Gene expression resultsRelative gene expression or expression ratio (R) was calculated for the hippocampus and the olfactory bulb. We performed pairwise comparisons of the three experi-mental conditions for each tissue. Mice placed in the tunnel (test group without filter cap or control group A in the tunnel with a filter cap) were compared to a control group (control B) that was kept in a building near the tun-nel. Finally, we also determined possible differences in gene expression between both groups that were placed in the tunnel (test group versus control A with filter cap). The results mentioned in Figures 4 and 5 are the expres-sion ratios (R) of the target genes for hippocampal tissue and olfactory bulb tissue, respectively. Additionally, the expression ratio (R), confidence interval (CI 95%) and the significance value (P) for the genes of which the expres-sion was significantly changed in one or more conditions is given in Table 2. Significant changes were found for 4 out of 11 assays tested in the hippocampus and 5 out of 12 assays in the olfactory bulb and this was observed for one or more pairwise comparisons. The bars in Figures 4 and 5 reflect the expression ratios R for the three pairwise comparisons. The statistical significance can be inter-preted for each bar separately as it is the result of inde-pendent pairwise comparisons.

In hippocampal tissue, the expression of all four affected genes was upregulated (Figure 4). The expres-sion levels of COX2 (R = 1.69; p < 0.01), NOS2 (R = 1.75; p < 0.05), and NFE2L2 (R = 1.36; p < 0.05) were signifi-cantly upregulated in the test group compared to control

B. This can be seen in the black bars of the respective genes in Figure 4. The gene COX2 was significantly upregulated (R = 1.27; p < 0.05) in control A versus control B (white bar for COX2 in Figure 4). Finally, the expression levels of COX2 (R = 1.33; p < 0.05), NOS2 (R = 1.38; p < 0.01), and NOS3 (R = 1.42; p < 0.05) were significantly upregulated in the test group compared to control A (grey bar in Figure 4).

In olfactory bulb tissue, five genes were significantly downregulated (Figure 5). We observed a significant downregulation of the levels of IL1α (R = 0.43; p < 0.001), COX2 (R = 0.49; p < 0.05), and IL6 (R = 0.18; p < 0.05) in the animals exposed in the tunnel (test group) compared to the animals that were kept in the building (control B). This difference is visualized in the black bars of Figure 5.

A significant downregulation of IL6 expression (R = 0.38; p < 0.05) was observed in animals protected with the filter cap (control A) compared to control B (white bar for IL6 in Figure 5). A final pairwise comparison revealed that the expression of IL1α (R = 0.53; p < 0.01), COX2 (R = 0.38; p < 0.001), NFE2L2 (R = 0.70; p < 0.05), and brain-derived neurotrophic factor transcript 4 (BDNF) transcript 4 (R = 0.63; p < 0.05) was significantly downregulated in the test group compared to control A (grey bars in Figure 5).

Discussion

An animal study was set up in the Craeybeckx tunnel in Belgium, a busy highway tunnel with predominantly heavy duty and diesel passenger cars. We found signifi-cant changes in the expression of genes coding for rel-evant inflammatory mediators in the brains of mice that were exposed for 5 days to realistic levels of traffic pol-lution. This occurred in the absence of any evidence of pulmonary or systemic inflammation.

Figure 3. Alveolar macrophages obtained by bronchoalveolar lavage from mice that remained for 5 days in the tunnel in a cage without filter cap (test group), with filter cap (control A), or mice that remained in an indoor location close to the tunnel (control B). Black carbon (BC) is indicated with arrows.

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

682 I. Bos et al.

Inhalation Toxicology

Figure 4. Hippocampal gene expression of the selected genes. Data indicate the expression in the test group in the tunnel relative to control B in the building (black bars), the expression in control A in the tunnel with a filter cap relative to control B (white bars) and the expression in the test group relative to control A (gray bars). N = 5 for each group. Statistical significance of the expression ratio is indicated (*p < 0.05, **p < 0.01) for the test group relative to control B, ($p < 0.05, $$p < 0.01) for control A relative to control B, (#p < 0.05, ##p < 0.01) for the test group relative to control A. Values for interleukin 6 (IL6) were below the detection limit. BDNF, brain-derived neurotrophic factor; COX2, cyclooxygenase 2; IL1α, interleukin 1α; IL1β, interleukin 1β; NOS2, inducible nitric oxide synthase; NOS3, endothelial nitric oxide synthase; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; TNF, tumor necrosis factor.

Figure 5. Gene expression of the selected genes in the olfactory bulb. Data indicate the expression in the test group in the tunnel relative to control B in the building (black bars), the expression in control A in the tunnel with a filter cap relative to control B (white bars) and the expression in the test group relative to control A (gray bars). N = 5 for each group. Statistical significance of the expression ratio is indicated (*p < 0.05, **p < 0.01, ***p < 0.001) for the test group relative to control B, ($p < 0.05, $$p < 0.01) for control A relative to control B, (#p < 0.05, ##p < 0.01, ###p < 0.001) for the test group relative to control A. BDNF, brain-derived neurotrophic factor; COX2, cyclooxygenase 2; IL1α, interleukin 1α; IL1β, interleukin 1β; IL6, interleukin 6; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; NOS2, inducible nitric oxide synthase; NOS3, endothelial nitric oxide synthase; TNF, tumor necrosis factor.

Table 2. Expression ratio (R), 95% confidence interval (CI 95%) and significance value (p) for the genes of which the expression was significantly changed in one or more conditions.

TissueGene

Test group vs. control B Control A vs. control B Test group vs. control AR CI 95% p R CI 95% p R CI 95% p

HippocampusCOX2 1.69 1.12–3.11 <0.01 1.27 0.92–2.16 <0.05 1.33 0.95–1.82 <0.05NOS2 1.75 0.96–2.81 <0.05 1.27 0.66–2.23 0.23 1.38 1.07–1.82 <0.01NOS3 1.59 0.68–3.61 0.16 1.12 0.43–2.65 0.7 1.42 0.94–2.36 <0.05NFE2L2 1.36 0.94–1.89 <0.05 1.26 0.58–2.49 0.34 1.08 0.56–2.27 0.72Olfactory bulb

IL1α 0.43 0.24–0.7 <0.001 0.82 0.40–1.37 0.26 0.53 0.31–0.97 <0.01

COX2 0.49 0.13–1.06 <0.05 1.31 0.34–2.67 0.47 0.38 0.23–0.61 <0.001NFE2L2 0.82 0.52–1.53 0.24 1.17 0.69–2.23 0.36 0.70 0.49–1.11 <0.05BDNF 4 0.6 0.26–1.41 0.08 0.94 0.49–1.74 0.76 0.63 0.34–1.18 <0.05IL6 0.18 0.05–1.01 <0.05 0.38 0.12–1.4 <0.05 0.46 0.15–2.16 0.08

BDNF 4, brain-derived neurotrophic factor transcript 4; COX2, cyclooxygenase 2; IL6, interleukin 6; IL1α, interleukin 1α; NOS2, inducible nitric oxide synthase; NOS3, endothelial nitric oxide synthase; NFE2L2, nuclear factor (erythroid-derived 2)-like 2.

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

Changed gene expression in mice brains 683

© 2012 Informa Healthcare USA, Inc.

The air quality was intensively analyzed during the experiment. We have found that mice in the tunnel were exposed to high levels of air pollution compared to controls (PNC 150 ± 25 103 cm−3, 55.1 μg PM2.5/m3, 23.1 μg OC/m3, and 13.9 μg EC/m3). BC ranged between 14 μg/m3 at night up to 150 μg/m3 during peak hours. PM concentrations that are much higher than those com-mon in ambient air in cities are often necessary to induce measurable effects in short-term clinical experimental studies. In addition, inhalation toxicity studies typically involve discontinuous exposure implicitly assuming that there is no dose-rate effect. Furthermore, many animal studies have the disadvantage that either the concen-tration or the composition of air pollution may not be representative of the conditions routinely encountered by humans. By using the tunnel as the exposure environ-ment for an in vivo study we could circumvent many of these problems.

After 5 days of exposure, carbon particles were abun-dantly present in alveolar macrophages of animals of the test group, and absent in control A and B. This indi-cates that the exposure to air pollution inside the tun-nel resulted in a relatively high internal dose of black carbon (BC) particles, while the filter cage of control A protected the animals from being exposed to BC. Despite the fact that mice in this study were exposed to relatively high concentrations of PM2.5, UFP, and BC, very few significant results emerged. There were no histological or physiological signs of pulmonary inflammation in the group exposed to tunnel air compared to the con-trol groups. None of the parameters measured in blood or BAL showed a relationship with PM exposure. Our experiment indicates that the duration of exposure prob-ably needs to be longer before significant adverse effects (a pulmonary or systemic inflammatory response) may manifest themselves.

Although, no physiological or histological inflamma-tory response could be detected, significant gene expres-sion changes in brain tissues were observed in response to PM exposure in the tunnel (analyzed by comparing the test group with control A). Gene expression analysis has proven to be a feasible alternative to traditional analysis in detecting early responses to exposure (van Berlo et al., 2010). We have analyzed the bulbus olfactorius because it is suspected to be the prime target of ultrafine particles when migrating through the olfactory nerve (Oberdörster et al., 2004; Block & Calderón-Garcidueñas, 2009). The hippocampal area of the brain was selected because it is a very sensitive region that is involved in learning and memory (Vaynman et al., 2004). We observed opposite responses in the hippocampus and the olfactory bulb, with mainly a gene expression upregulation in the hip-pocampus and a downregulation in the olfactory bulb in response to air pollution exposure. Previously, van Berlo et al., 2010 also found that short-term inhalation expo-sure to diesel engine exhaust triggered region-specific gene expression changes in rat brain (van Berlo et al., 2010; Gerlofs-Nijland et al., 2010).

In the hippocampus, PM exposure induced a signifi-cant increase in gene expression of inflammatory markers COX2, NOS2, and NOS3. The mice that were placed in the tunnel with a filtercap (control A) also showed increased COX2 expression in the hippocampus compared to con-trol B, however this increase was significantly smaller compared to the test group. The increased expression of inflammatory markers in the hippocampus is consistent with literature showing that chronic exposure to air pollu-tion induces neuroinflammation. Previously, Calderón-Garcidueñas et al., 2008a have shown increases in the expression of COX2 and IL1β in the olfactory bulb, fron-tal cortex, substantia nigra and vagus nerves of healthy children and young adults living in Mexico City who died suddenly. However, they did not find any expression changes in the hippocampus. Calderón-Garcidueñas et al., 2008b showed increases in COX2 expression in frontal white matter of Mexico City dogs. Campbell et al., 2005 detected increased levels of IL1α and TNFα in brain tissue of mice exposed to particulate matter for two weeks. They also found increases in the level of immune related NFkB. NFkB activation is known to activate the transcription of NOS2 (Mohankumar et al., 2008). The test group showed increased expression of NFE2L2, a tran-scription factor that activates the antioxidant response, compared to control B. Increased oxidative stress in the cell induces a protective, antioxidant response via activa-tion of NFE2L2 which in its turn activates transcription of multiple, antioxidant genes as well as its own expression (Ishii et al., 2000; Nguyen et al., 2003; Stewart et al., 2003). Air pollution is known to increase oxidative stress, also in the brain (Mohankumar et al., 2008). We suggest that increased NFE2L2 expression in the test group may be a protective response to increased oxidative stress induced by air pollution exposure.

In the olfactory bulb, PM exposure was associated with a downregulation of two inflammatory markers IL1α, COX2, as well as a downregulation of NFE2L2 and BDNF transcript 4. In mice, the expression of the neurotrophine BDNF is controlled by different promoters (depending on tissue type and activity) resulting in the transcription of 4 different mRNA’s (Aid et al., 2007). Since nothing is known about the expression of the different transcripts in the brain tissues collected from C57Bl6 mice, we studied the expression of the 4 transcripts. IL6 was downregu-lated in the test group as well as control A, suggesting an effect of the tunnel rather than PM exposure. Previous studies suggest that the olfactory bulb may be the prime target of the central nervous system. It was shown that particles can migrate directly from the deposition on the nasal epithelium to the olfactory bulb (Oberdörster et al., 2004). IL1α and COX2 are genes that are normally upregulated with inflammation. We hypothesized that the expression of these genes would increase based on previous findings of Calderón-Garcidueñas and col-leagues, who detected increased levels of COX2 and IL1β mRNA in the olfactory bulb of humans living in heavy polluted Mexico City (Calderón-Garcidueñas

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

684 I. Bos et al.

Inhalation Toxicology

et al., 2004, 2007, 2008a, 2011). Calderón-Garcidueñas and colleagues measured the effect of long-term expo-sure to air pollution of Mexico City in humans and dogs. The two major components of the air pollution in Mexico City are PM and ozone, a reactive oxygen species also linked to inflammation and neuropathology (Block & Calderón-Garcidueñas, 2009; Calderón-Garcidueñas et al., 2004, 2007, 2008a, 2011). This is in contrast to our study in which we measured short-term effects of expo-sure to traffic-related air pollution in mice that were kept in a tunnel. Also short-term studies in rodents have found an induction of neuroinflammation in response to PM exposure (Campbell et al., 2005; 2009; Kleinman et al., 2008; Tin-Tin-Win-Shwe et al., 2006). However, the design of these studies is also different from our design. Some studies have analyzed the effect of PM exposure on whole brain tissue or cortex of rodents (Campbell et al., 2005; 2009; Kleinman et al., 2008). Tin-Tin-Win-Shwe and colleagues intranasally instilled carbon black and detected increased IL1β, TNFα, and chemokine mRNA levels in the olfactory bulb and not in the hippocampus (Tin-Tin-Win-Shwe et al., 2006). They demonstrated for the first time a region-specific brain cytokine and che-mokine mRNA induction in mice. In our study, we also find a region-specific response, however with an overall upregulation of inflammatory gene expression in the hippocampus and a downregulation of inflammatory gene expression in the olfactory bulb in response to traffic-related air pollution exposure. In accordance to our study, Gerlofs-Nijland et al. investigated the effect of short term exposure (i.e. 4 weeks) to diesel engine exhaust on different brain areas. They also detected a region-specific response with a decreased TNFα level in the olfactory bulb and the tubercles, an increased TNFα level in the striatum, and no changes for TNFα in the cortex, hippocampus and cerebellum (Gerlofs-Nijland et al., 2010). In addition, the level of IL1α was also only increased in the striatum. They suggested that the region-specific response may be related to the unique cellular and molecular composition and function of dif-ferent brain regions (Gerlofs-Nijland et al., 2010). Thus, although increased levels of inflammatory markers are found in whole brain tissue of mice, our study in addition to Tin-Tin-Win-Shwe et al., 2006; Gerlofs-Nijland et al., 2010 shows a region-specific response after a short-term PM exposure. However, more research is needed to ana-lyze the effect of the composition, duration, and mode of the exposure on the response in different brain areas, and the pathways leading to neuroinflammation and neuropathology. In our study, COX2 gene expression was altered in both hippocampus and olfactory bulb and this suggests that COX2 is an important molecule in medi-ating effects of PM. PM exposure was associated with a down regulation of BDNF transcript 4 in the olfactory bulb. We suggest that a downregulation of BDNF gene expression may result in lower protein levels of BDNF which in turn may lead to less neurotrophic support. In humans, BDNF levels increase acutely in response to an

exercise bout (Rojas Vega et al., 2006; Ferris et al., 2007; Goekint et al., 2011; Griffin et al., 2011). It is suggested that exercise increases BDNF levels by increasing pro-duction and release from the brain (Rasmussen et al., 2009). Recently, our group has shown in a human inter-vention study that although the BDNF concentration in serum increased after cycling in an air-filtered room, it did not increase when healthy participants performed the same cycling test along a busy traffic road (Bos et al., 2011). The decreased BDNF expression in this study adds evidence to our hypothesis on the effect of air pollution on neurotrophine expression.

Tunnel exposure without PM, also affected gene expression, evidenced by increased COX2 expression in the hippocampus and decreased IL6 expression in the olfactory bulb (control A compared to control B). The cause may be that the filter cap did not filter all particles or gases. Also, we cannot exclude that stress caused by noise inside the tunnel may have influenced gene expres-sion. However, by comparing the test group (tunnel + PM) with the control A (tunnel without PM) we were able to exclude potential confounders (such as stress) coupled to the tunnel exposure from the effects of PM exposure.

Our set-up allows discriminating between the effect of being inside the tunnel (including exposure to noise and stress) and the effects of exposure to fresh traffic-related air pollution. This was accomplished by using two controls; one in the tunnel but shielded from par-ticulate air pollution (control A) and another placed in a building near the tunnel (control B). Many studies have used intra-tracheal (e.g. Happo et al., 2010) or intra-nasal (Tellabati et al., 2010) instillation of high doses of col-lected PM to study toxic effects. There are however clear limitations to using intra-tracheal instillation including: bypass of upper respiratory tract, immediate exposure, less homogeneous distribution of the particles in the lung (Driscoll et al., 2000). The latter may result in dif-ferences in clearance pathways, doses to certain cells, degree, and site of systemic absorption compared to inhalation studies. Intra-tracheal instillation studies are therefore less suited for deriving a dose-response curve for inhalation of particles. A potential weakness of the study presented here was that stress (due to the condi-tions in the tunnel esp. noise) could have been a factor in the response. Clougherty et al., 2010 found a significantly greater increase in respiratory frequency and a greater decrease in many respiratory parameters in stressed animals compared with non-stressed animals. These authors observed changes in biomarkers indicating sys-temic inflammation responses associated with airway disease among animals exposed to both stress and par-ticles, 5 hour/day for 10 days. By placing control groups inside and outside the tunnel, the effects of stress and air pollution could be separated, but future experiments should include measurements of stress hormones. This study shows a tissue-specific response to a short-term PM exposure in a tunnel environment with increased COX2, NOS2, and NOS3 mRNA levels in the hippocampus and

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

Changed gene expression in mice brains 685

© 2012 Informa Healthcare USA, Inc.

decreased IL1α, COX2, NFE2L2, and BDNF mRNA levels in the olfactory bulb. Although these observations sug-gest an effect of PM exposure, similar changes need to arise at protein level before functional effects can occur. Future research needs to focus on confirming our find-ings on protein levels.

Conclusions

The design of our study allowed investigating real and relevant particles (at realistic high doses) and at the same time control for effects of well-characterized co-existing factors in the environment. Our study shows that the use of a tunnel is a feasible and a strategically relevant mode for studying health effects of PM exposure. In addition, the study provided evidence of the presence of traffic-related particles in the lungs of mice placed in the tunnel without filters. An exposure of 5 days did not induce any histo-pathological or toxic effects. However, we found that gene expression in the brain is affected by exposure to PM. We observed a region-specific response with opposite effects in different brain regions, with an increase of inflamma-tory gene expression in the hippocampus, and a decreased gene expression of proteins functional in inflammation, and neurotrophic support in the olfactory bulb.

Acknowledgments

The authors wish to thank the Flemish Traffic Control center for the help in locating animals and instruments in and outside the tunnel. We acknowledge Wesley Dubois and Karen Hollanders for technical support in the gene expression analysis. Finally, we appreciate Lotte Jacobs for her help with the study design, performing the experi-ment and data analysis.

Declaration of interest

The authors declare that they have no competing inter-ests. This study was partly financed by a strategic research grant of the Flemish government agency for Innovation by Science and Technology to the Particulair project proposal, a VITO PhD scholarship to Ms. Inge Bos, and VITO strategic research funds.

ReferencesAid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T. 2007. Mouse and

rat BDNF gene structure and expression revisited. J Neurosci Res 85:525–535.

Bos I, Jacobs L, Nawrot TS, de Geus B, Torfs R, Int Panis L, Degraeuwe B, Meeusen R. 2011. No exercise-induced increase in serum BDNF after cycling near a major traffic road. Neurosci Lett 500:129–132.

Block ML, Calderón-Garcidueñas L. 2009. Air pollution: mechanisms of neuroinflammation and CNS disease. Trends Neurosci 32:506–516.

Brook RD, Rajagopalan S, Pope CA 3rd, Brook JR, Bhatnagar A, Diez-Roux AV, Holguin F, Hong Y, Luepker RV, Mittleman MA, Peters A, Siscovick D, Smith SC Jr, Whitsel L, Kaufman JD; American Heart Association Council on Epidemiology and Prevention, Council on

the Kidney in Cardiovascular Disease, and Council on Nutrition, Physical Activity and Metabolism. 2010. Particulate matter air pollution and cardiovascular disease: An update to the scientific statement from the American Heart Association. Circulation 121:2331–2378.

Campbell A, Oldham M, Becaria A, Bondy SC, Meacher D, Sioutas C, Misra C, Mendez LB, Kleinman M. 2005. Particulate matter in polluted air may increase biomarkers of inflammation in mouse brain. Neurotoxicology 26:133–140.

Campbell A, Araujo JA, Li H, Sioutas C, Kleinman M. 2009. Particulate matter induced enhancement of inflammatory markers in the brains of apolipoprotein E knockout mice. J Nanosci Nanotechnol 9:5099–5104.

Calderón-Garcidueñas L, Reed W, Maronpot RR, Henríquez-Roldán C, Delgado-Chavez R, Calderón-Garcidueñas A, Dragustinovis I, Franco-Lira M, Aragón-Flores M, Solt AC, Altenburg M, Torres-Jardón R, Swenberg JA. 2004. Brain inflammation and Alzheimer’s-like pathology in individuals exposed to severe air pollution. Toxicol Pathol 32:650–658.

Calderón-Garcidueñas L, Franco-Lira M, Torres-Jardón R, Henriquez-Roldán C, Barragán-Mejía G, Valencia-Salazar G, González-Maciel A, Reynoso-Robles R, Villarreal-Calderón R, Reed W. 2007. Pediatric respiratory and systemic effects of chronic air pollution exposure: nose, lung, heart, and brain pathology. Toxicol Pathol 35:154–162.

Calderón-Garcidueñas L, Solt AC, Henríquez-Roldán C, Torres-Jardón R, Nuse B, Herritt L, Villarreal-Calderón R, Osnaya N, Stone I, García R, Brooks DM, González-Maciel A, Reynoso-Robles R, Delgado-Chávez R, Reed W. 2008a. Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults. Toxicol Pathol 36:289–310.

Calderón-Garcidueñas L, Mora-Tiscareño A, Ontiveros E, Gómez-Garza G, Barragán-Mejía G, Broadway J, Chapman S, Valencia-Salazar G, Jewells V, Maronpot RR, Henríquez-Roldán C, Pérez-Guillé B, Torres-Jardón R, Herrit L, Brooks D, Osnaya-Brizuela N, Monroy ME, González-Maciel A, Reynoso-Robles R, Villarreal-Calderon R, Solt AC, Engle RW. 2008b. Air pollution, cognitive deficits and brain abnormalities: a pilot study with children and dogs. Brain Cogn 68:117–127.

Calderón-Garcidueñas L, Engle R, Mora-Tiscareño A, Styner M, Gómez-Garza G, Zhu H, Jewells V, Torres-Jardón R, Romero L, Monroy-Acosta ME, Bryant C, González-González LO, Medina-Cortina H, D’Angiulli A. 2011. Exposure to severe urban air pollution influences cognitive outcomes, brain volume and systemic inflammation in clinically healthy children. Brain Cogn 77:345–355.

Chen JC, Schwartz J. 2009. Neurobehavioral effects of ambient air pollution on cognitive performance in US adults. Neurotoxicology 30:231–239.

Clougherty JE, Rossi CA, Lawrence J, Long MS, Diaz EA, Lim RH, McEwen B, Koutrakis P, Godleski JJ. 2010. Chronic social stress and susceptibility to concentrated ambient fine particles in rats. Environ Health Perspect 118:769–775.

Driscoll KE, Costa DL, Hatch G, Henderson R, Oberdorster G, Salem H, Schlesinger RB. 2000. Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations. Toxicol Sci 55:24–35.

Ferris LT, Williams JS, Shen CL. 2007. The effect of acute exercise on serum brain-derived neurotrophic factor levels and cognitive function. Med Sci Sports Exerc 39:728–734.

Gerlofs-Nijland ME, van Berlo D, Cassee FR, Schins RP, Wang K, Campbell A. 2010. Effect of prolonged exposure to diesel engine exhaust on proinflammatory markers in different regions of the rat brain. Part Fibre Toxicol 7:12.

Gertler AW, Abu-Allaban M, Coulombe W, Gillies JA, Pierson WP, Rogers FC, Sagebiel JC, Tarnay L, Cahill TA. 2001. Measurements

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.

686 I. Bos et al.

Inhalation Toxicology

of mobile source particulate emissions in a highway tunnel. Int J Vehicle Des 27:86–93.

Goekint M, Roelands B, Heyman E, Njemini R, Meeusen R. 2011. Influence of citalopram and environmental temperature on exercise-induced changes in BDNF. Neurosci Lett 494:150–154.

Griffin ÉW, Mullally S, Foley C, Warmington SA, O’Mara SM, Kelly AM. 2011. Aerobic exercise improves hippocampal function and increases BDNF in the serum of young adult males. Physiol Behav 104:934–941.

Happo MS, Salonen RO, Hälinen AI, Jalava PI, Pennanen AS, Dormans JA, Gerlofs-Nijland ME, Cassee FR, Kosma VM, Sillanpää M, Hillamo R, Hirvonen MR. 2010. Inflammation and tissue damage in mouse lung by single and repeated dosing of urban air coarse and fine particles collected from six European cities. Inhal Toxicol 22:402–416.

Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, Yamamoto M. 2000. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 275:16023–16029.

Jacobs L, Emmerechts J, Mathieu C, Hoylaerts MF, Fierens F, Hoet PH, Nemery B, Nawrot TS. 2010a. Air pollution related prothrombotic changes in persons with diabetes. Environ Health Perspect 118:191–196.

Jacobs L, Nawrot TS, de Geus B, Meeusen R, Degraeuwe B, Bernard A, Sughis M, Nemery B, Panis LI. 2010b. Subclinical responses in healthy cyclists briefly exposed to traffic-related air pollution: an intervention study. Environ Health 9:64.

Kleinman MT, Araujo JA, Nel A, Sioutas C, Campbell A, Cong PQ, Li H, Bondy SC. 2008. Inhaled ultrafine particulate matter affects CNS inflammatory processes and may act via MAP kinase signaling pathways. Toxicol Lett 178:127–130.

MohanKumar SM, Campbell A, Block M, Veronesi B. 2008. Particulate matter, oxidative stress and neurotoxicity. Neurotoxicology 29:479–488.

Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB. 2003. Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome. J Biol Chem 278:4536–4541.

Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W, Cox C. 2004. Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 16:437–445.

Peterson MR, Richards MH. 2002. Thermal-optical-transmittance analysis for organic, elemental, carbonate, total carbon, and OCX2 in PM2.5 by the EPA/NIOSH method. In: Winegar ED, Tropp RJ, ed. Proceedings, Symposium on Air Quality Measurement Methods and Technology. Pittsburgh, PA: Air & Waste Management Association, 83-1–83-19.

Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36.

Phuleria HC, Geller MD, Fine PM, Sioutas C. 2006. Size-resolved emissions of organic tracers from light- and heavy-duty vehicles measured in a California roadway tunnel. Environ Sci Technol 40:4109–4118.

Pope CA 3rd, Ezzati M, Dockery DW. 2009. Fine-particulate air pollution and life expectancy in the United States. N Engl J Med 360:376–386.

Ranft U, Schikowski T, Sugiri D, Krutmann J, Krämer U. 2009. Long-term exposure to traffic-related particulate matter impairs cognitive function in the elderly. Environ Res 109:1004–1011.

Rasmussen R. 2001. Quantification on the LightCycler. In: Meuer S, Wittwer C, Nakagawara K, eds. Rapid cycle real-time PCR methods and applications. Heidelberg: Springer Press, 21–34.

Rasmussen P, Brassard P, Adser H, Pedersen MV, Leick L, Hart E, Secher NH, Pedersen BK, Pilegaard H. 2009. Evidence for a release of brain-derived neurotrophic factor from the brain during exercise. Exp Physiol 94:1062–1069.

Rojas Vega S, Strüder HK, Vera Wahrmann B, Schmidt A, Bloch W, Hollmann W. 2006. Acute BDNF and cortisol response to low intensity exercise and following ramp incremental exercise to exhaustion in humans. Brain Res 1121:59–65.

Stewart D, Killeen E, Naquin R, Alam S, Alam J. 2003. Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium. J Biol Chem 278:2396–2402.

Sturm PJ, Rodler J, Lechner B, Almbauer RA. 2001. Validation of emission factors for road vehicles based on street tunnel measurements. Int J Vehicle Des 27:65–75.

Suglia SF, Gryparis A, Wright RO, Schwartz J, Wright RJ. 2008. Association of black carbon with cognition among children in a prospective birth cohort study. Am J Epidemiol 167:280–286.

Tellabati A, Fernandes VE, Teichert F, Singh R, Rylance J, Gordon S, Andrew PW, Grigg J. 2010. Acute exposure of mice to high-dose ultrafine carbon black decreases susceptibility to pneumococcal pneumonia. Part Fibre Toxicol 7:30.

Tin-Tin-Win-Shwe, Yamamoto S, Ahmed S, Kakeyama M, Kobayashi T, Fujimaki H. 2006. Brain cytokine and chemokine mRNA expression in mice induced by intranasal instillation with ultrafine carbon black. Toxicol Lett 163:153–160.

van Berlo D, Albrecht C, Knaapen AM, Cassee FR, Gerlofs-Nijland ME, Kooter IM, Palomero-Gallagher N, Bidmon HJ, van Schooten FJ, Krutmann J, Schins RP. 2010. Comparative evaluation of the effects of short-term inhalation exposure to diesel engine exhaust on rat lung and brain. Arch Toxicol 84:553–562.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034.

Vaynman S, Ying Z, Gomez-Pinilla F. 2004. Hippocampal BDNF mediates the efficacy of exercise on synaptic plasticity and cognition. Eur J Neurosci 20:2580–2590.

Vercauteren J, Matheeussen C, Wauters E, Roekens E, van Grieken R, Krata A, Makarovska Y, Maenhaut W, Chi X, Geypens B. 2011. Chemkar PM10: An extensive look at the local differences in chemical composition of PM10 in Flanders Belgium. Atmospheric Env 45:108–116.

Weingartner E, Keller C, Stahel WA, Burtscher H, Baltensperger U. 1997. Aerosol emission in a road tunnel. Atmospheric Env 31:451–462.

Weuve J, Puett RC, Schwartz J, Yanosky JD, Laden F, Grodstein F. 2012. Exposure to particulate air pollution and cognitive decline in older women. Arch Intern Med 172:219–227.

Online supplementary materials are available at: http://informahealthcare.com/iht

Inha

latio

n T

oxic

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y V

ito o

n 08

/21/

12Fo

r pe

rson

al u

se o

nly.