Bitumen fume-induced gene expression profile in rat lung

Toxicology and Applied Pharmacology 215 (2006) 83 – 92 www.elsevier.com/locate/ytaap

Bitumen fume-induced gene expression profile in rat lung Laurent Gate ⁎, Cristina Langlais, Jean-Claude Micillino, Hervé Nunge, Marie-Claire Bottin, Richard Wrobel, Stéphane Binet Institut National de Recherche et Sécurité, Avenue de Bourgogne, BP 27, 54501 Vandoeuvre Cedex, France Received 29 November 2005; revised 26 January 2006; accepted 26 January 2006 Available online 10 March 2006

Abstract Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 °C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure. © 2006 Elsevier Inc. All rights reserved. Keywords: Bitumen fumes; Microarray; Polycyclic aromatic hydrocarbon; Inflammation; Inhalation

Introduction Bitumen fumes are a complex mixture of particles and vapors containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons (PAH). Occupational exposure to these fumes during road paving or roofing activities may represent a health risk (WHO, 2004), since exposure to free or particle-bound PAH has been associated with lung DNA mutation in exposed laboratory animals (Sato et al., 2000; Hashimoto et al., 2005) and other adverse health effects in several occupational and environmental conditions (McClellan, 1987). However, epidemiological studies failed to draw unambiguous conclusions concerning the adverse health effects, especially increased lung cancer incidence, due to

⁎ Corresponding author. Fax: +33 3 83 50 98 46. E-mail address: [email protected] (L. Gate). 0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2006.01.012

bitumen fumes exposure (Melius, 2003). Similarly, discrepant results were obtained during biomonitoring studies; for example, a study reported sister-chromatide exchange and micronuclei formation in peripheral lymphocytes from road pavers (Burgaz et al., 1998), while in another similar work, such genetic alterations were not observed (Jarvholm et al., 1999). Such differences might be explained by the complex nature of bitumen, indeed asphalt content in PAH and other toxic compounds may greatly vary from one batch to another based on its origin (WHO, 2004). In addition, epidemiological studies on the occurrence of non-malignant lung diseases among bitumen workers have shown an increased risk of respiratory symptoms, lung function decline, and chronic obstructive pulmonary deficiency among these workers compared to their construction counterparts (Randem et al., 2004). In vivo cellular effects of bitumen fumes have been usually studied in regard to their content in PAH. Carcinogenic PAH such as benzo[a]pyrene (BaP) or 7,12-dimethylbenz(a)anthracene

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(DMBA), bind and activate the nuclear receptor aryl hydrocarbon receptor (AhR) (Rowlands and Gustafsson, 1997) which induced the expression of many genes including the cytochrome P450 1A1 (CYP1A1), aldehyde reductase 3A1 (ALDH3A1) and NAD(P)H:quinone oxidoreductase (NQO1). Increased expression and activity of CYP1A1 and NQO1 have been detected in lung from rats exposed to bitumen fumes (inhalation) or condensates (skin painting) and were associated with DNA adducts formation (Ma et al., 2002, 2003; Wang et al., 2003). CYP1A1 metabolizes PAH into reactive PAH-dihydrodiolepoxide metabolites which can react with DNA bases. The formation of DNA adducts may play a critical role in the first step of mutagenesis and is believed to be involved in the carcinogenic potential of PAH (Miller and Ramos, 2001; Galvan et al., 2005). Oligonucleotide and cDNA microarray technologies, which allow to analyze the expression of tens of thousands of genes at once, have been extensively used in the past few years to characterize the mechanisms of action of various xenobiotics including hepatic (Hamadeh et al., 2002; Waring et al., 2003) and pulmonary toxicants (Shultz et al., 2004; Dillman et al., 2005). Based on their high throughput capabilities, many authors have also used microarrays to attempt to characterize xenobiotic toxicological profiles on the basis of their transcriptional signatures (Burczynski et al., 2000; Ellinger-Ziegelbauer et al., 2005). Through the development of databases and the standardization of microarray techniques (Mattes et al., 2004), this technology may become a valuable predictive tool for determining the mechanisms associated with the toxicity of a given compound. Although previous studies have demonstrated the role of PAH in bitumen fumes toxicity, they gave an incomplete representation of the pulmonary mechanism of action of such aerosols. In order to gain additional insights on the biological impact of asphalt fumes exposure, we used oligonucleotide microarrays to identify new pathways affected by this complex mixture. Methods Exposure design and bitumen fume characterization. A 50/70 pen batch of bitumen (CAS No: 8052-42-4) from Venezuela was used for this experiment. The experimental device used for bitumen fume generation and nose-only animal exposition has been described previously (Binet et al., 2002a). Briefly, the fume generator was made of a 10 L bitumen vessel and a fume chamber. The bitumen vessel was heated at 170 °C with an electric hot plate, the target atmospheric concentration in Total Particulate Matter (TPM) was 100 mg/m3. Homogeneous bitumen mixture was obtained by stirring the vessel during the generation period. Compressed and filtered air (Ultrafilter International, Germany) was admitted horizontally into a fume chamber at constant flow rate (23 L/min) and temperature (20 °C) with 40% of relative humidity. The air flow containing the bitumen fumes was introduced at the top of a 37 L inhalation chamber and exhausted at the bottom. The front door included several ports for sampling the chamber's atmosphere and connecting plethysmographs used for animal nose-only exposure. The PAH profile of the bitumen fumes generated using our device was comparable to that measured during road paving operations (Binet et al., 2002a). Animals and experimental design. Two-month-old Fisher 344 male rats weighing about 180 g were purchased from Charles River (Les Oncins, France). The animals were husbanded at the INRS laboratory animal facility licensed by

the French Agriculture Department (agreement #A54550) into polycarbonate cages (1/cage) covered with spun-bonded polyester cage filter and fed with pellet food and water ad libitum. Room temperature was 21 ± 1 °C, air pressure was 5 mm H2O above the atmospheric pressure, humidity ranged from 40 to 60% and lighting was on for 12 h/day. Rats (n = 5) were exposed to bitumen fumes for 5 days, 6 h per day in a nose-only inhalation chamber as described previously (Binet et al., 2002a). Control animals (n = 5) were exposed in similar conditions to clean air. Animals were terminated immediately at the end of the last day of exposure. Lung collection. Lungs and trachea were dissected, the left lung was ligatured, removed, placed in a tube containing 20 mL of RNAlater (Ambion, Huntingdon, UK), stored at 4 °C overnight and then at −20 °C until RNA extraction. Bronchoalveolar lavages (BAL) were performed on the remaining right lung using 2 × 4 mL of PBS, pH 7.4. BAL were centrifuged 10 min at 400 × g, the cell pellets were resuspended in 1 mL of ice cold PBS, 100 μL of cell suspension was used for cytospin preparation and May-Grunwald Giemsa staining. The remaining cells were centrifuged, the resulting pellets were resuspended in the lysis buffer for RNA extraction (Stratagene), flash-frozen in liquid nitrogen and stored at −80 °C. Right lungs were also flash-frozen in liquid nitrogen and stored at −80 °C. RNA extraction. Total RNA from left lung tissue was extracted using Qiagen RNeasy Midi kit (Qiagen, Courtaboeuf, France) following the manufacturer's instructions. RNA samples were subsequently repurified using Stratagene Absolutely RNA RT-PCR Miniprep kit including the DNAse I digestion step according to the manufacturer's instructions (Stratagene, Amsterdam, The Netherlands). RNA quality and concentration were measured using an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA from BAL cells were extracted using Stratagene Absolutely RNA RT-PCR Miniprep kit including the DNAse I digestion step according to the manufacturer's instructions. RNA quality was determined on a 1.5% agarose gel and concentrations were measured using a Beckman DU 640 B spectrophotometer (Beckman Coulter, Villepinte, France). Total RNA amplification and labeling, and microarray data extraction. RNA from the control animals (n = 5) was pooled in equal amounts, while RNA from the exposed animals (n = 5) was analyzed individually. Each treated sample was compared to the pooled control, dye swaps were also performed. 500 ng of RNA from pooled control or individual exposed animals was amplified in the presence of 24 nmol of Cyanine 3-CTP or Cyanine 5-CTP (PerkinElmer, Boston, MA) using the Agilent Low Fluorescent Linear Amplification kit according to the manufacturer's instructions. The excesses of flurochromes were removed using the Qiagen RNeasy Mini kit. The amounts of purified cRNA and incorporated Cyanine dyes were measured using a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE). Each labeled cRNA (750 ng) was used for hybridization on Agilent 22K Rat Oligo Microarray slides according to the Agilent 60-mer oligo microarray processing protocol. After a 15-h hybridization at 60 °C in an Agilent hybridization oven, slides were washed following the SSPE wash procedure described in the Agilent user's manual instructions. Slides were then read using an Agilent DNA microarray scanner and data were extracted and normalized using the default parameters of the Agilent's Feature Extraction software v7.5. Microarray data analysis. Normalized data were analyzed using Rosetta Luminator v3.0 (Rosetta Biosoftware, Kirkland, WA). Genes with an intensity ratio statistically significant in each experiment (P < 0.05) were considered as differentially expressed. We map the differentially expressed (DE) probe set to Gene Ontology (GO) (Beissbarth and Speed, 2004) in order to perform a more advanced analysis of the pulmonary modifications induced by bitumen fumes. The Web-based software GOstat (http://www.gostat.wehi.edu.au) (Beissbarth and Speed, 2004) allows to determine the significantly (Benjamini and Hochberg false discovery rate P < 0.1) over-represented Gene Ontology Biological Process, Molecular Function and Cellular Component from the DE probes set by comparison with the probes spotted on the microarray slide (reference group). The software

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Table 1 Statistically over-represented GO categories within the Biological process node GO ID

GO terms

Number changed

Number detected

Number in the group

P value

GO:0008150 GO:0006950 GO:0006952 GO:0009611 GO:0009607 GO:0009613 GO:0006955 GO:0043207 GO:0050896 GO:0006968 GO:0009605 GO:0044248 GO:0030595 GO:0006954 GO:0042102

Biological process Response to stress Defense response Response to wounding Response to biotic stimulus Response to pest, pathogen or parasite Immune response Response to external biotic stimulus Response to stimulus Cellular defense response Response to external stimulus Cellular catabolism Immune cell chemotaxis Inflammatory response Positive regulation of T-cell proliferation

23 17 13 19 12 16 12 30 4 19 21 3 6 2

235 174 93 211 83 165 88 432 10 248 291 7 41 3

501 539 153 633 157 481 167 1151 10 514 1659 7 79 3

0.000122 0.00254 0.00254 0.00254 0.00254 0.00254 0.0033 0.00563 0.0122 0.0242 0.0309 0.048 0.077 0.0923

counts the number of appearances of each GO term associated with genes from the DE probe list and from the reference group. For each GO term, a P value is calculated representing the probability that the observed numbers of counts result from the random distribution of this GO term between the DE genes group and the reference group. Further investigations were performed using GenMAPP v2.0 software (http://www.genmapp.org) (Dahlquist et al., 2002; Doniger et al., 2003), which allows to analyze microarray pathway profiles (MAPPs) representing biological pathways and identify significantly over-represented GenMAPPs and KEGG pathways associated with the probe set altered by bitumen fumes (P < 0.05). All the data analyses were performed by using as gene identifier the Unigene ID of the genes and ESTs from the DE probe set and the reference group. RNA reverse transcription and quantitative PCR (qPCR). Total RNA (500 ng) from lung tissue or BAL cells was reverse-transcribed with 500 ng of oligo (dT)12–18 using the SuperScript II RT (50 U) (Invitrogen) following the manufacturer's protocol. As a negative control, a sample containing RNA but without RT enzyme was also included. QPCR was performed with a LightCycler (Roche, Meylan, France) using the QuantiTect SYBR Green PCR kit (Qiagen). Briefly, 25 ng of reversetranscribed RNA was mixed with 5 pmol of specific primers for a given gene and the QuantiTect SYBR Green PCR mix. PCR amplification was carried out as follow: 15 min at 95 °C; n cycles [15 s at 95 °C, 20 s at x °C and 15 s at 72 °C]; where n and x depend on the primer set and the gene. Relative amount of each gene was determined by using the Pfaffl model (Pfaffl, 2001). For each gene, a standard curve was made and the subsequent slope was used to calculate the efficiency (E) of the PCR reaction (E = 10[−1/slope]). For each sample, the relative expression of a given gene was calculated from the threshold cycle (CT) value, which is the

number of cycles for which an increase in PCR product is first detected at a statistically significant level. Fold change ¼ ½ðEtarget ÞDCT target ðcontrol−exposedÞ
=½ðEbactin ÞDCT bactin ðcontrol−exposedÞ
The fold change of a target gene is expressed in the exposed animals vs. the control in comparison to βactin (used as a reference). Etarget is the real-time PCR efficiency of the target gene transcript; Eβactin is the real-time PCR efficiency of the βactin transcript.

Results Bitumen fume characterization Rats were exposed for 5 consecutive days, 6 h/day to bitumen fumes. The total particulate matter in the generated fumes was 114.6 ± 16.8 mg/m3. The mass median aerodynamic diameter of the particles was 2.6 ± 0.1 μm. Oligonucleotide microarray data analysis Microarray data analysis showed that following normalization and statistical analysis, 363 out of the 20500 probes available on the slide were differentially expressed in each exposed animal lung (P < 0.05). Using their Unigene ID as gene identifier, probes were annotated and clustered with the Gene

Table 2 Statistically over-represented GO categories within the molecular function and cellular component nodes GO ID

GO terms

GO:0003674 GO:0008009 GO:0019865 GO:0001664 GO:0004089 GO:0042708 GO:0008131 GO:0004175 GO:0004866 GO:0005575 GO:0005764

Molecular function Chemokine activity Immunoglobulin binding G-protein-coupled receptor binding Carbonate dehydratase activity Elastase activity Amine oxidase Endopeptidase activity Endopeptidase activity inhibitor Cellular component Lysosome

Number changed

Number detected

Number in the group

P value

5 3 5 3 2 2 13 6

21 7 26 8 3 3 166 56

37 8 43 25 3 3 800 238

0.0429 0.0633 0.0775 0.0775 0.0923 0.0923 0.0956 0.0959

6

36

48

0.0633

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Table 3 Significantly over-represented GenMAPPs associated with the bitumen fumeinduced gene list MAPP name

Number changed

Rn_Oxidative_tress 8 Rn_Nucleotide_GPCR 3

Number Number in measured the pathway

Z Permute score P value

27 7

4.682 <0.001 2.595 0.04

29 8

Ontology Web-based software GOstat. Within the DE probe set, 325 sequences were considered as unique genes and 160 as annotated genes; while in the full probe list, among the 20,500 probes, 13,656 were characterized as unique genes and 4523 as annotated genes. Significantly over-represented GO categories within Biological process, Molecular function and Cellular component nodes are presented in Tables 1 and 2. Further analyses were performed with GenMAPP 2.0 software which allowed to identify over-represented pathways available from the “contributed” MAPP Archive. DE and full probe lists were compared with a R. norvegicus gene database (Rn-Std_20050713_beta.gdb) provided with the software suite. Among the DE and full gene lists, 223 and 4068 were linked to the local MAPPs, respectively. Results generated by this software could be found in Table 3. On the basis of these data, the modulation of some biological mechanisms could be highlighted.

Fig. 1. Cytokines and chemokines expression in bronchoalveolar lavage cells from control and bitumen-exposed animals. Total RNA was extracted from BAL cells and the expression of TNFα, IL1β and MIP2 was determined by quantitative RT-PCR as described in Methods. *Significantly different from the control (Student's t test P < 0.05).

Bitumen fume-induced pulmonary inflammatory response As shown in Table 4, the total cell number in BAL from exposed animals was about twice that observed in the control group; significant increases in numbers of macrophages, neutrophilic granulocytes and lymphocytes were also detected. In addition, granulocyte percentages were higher in treated rats (19.3 ± 9.7 vs. 1.3 ± 0.6% in exposed and control groups, respectively), while percentages of macrophages were lower in exposed rodents as compared to controls. As demonstrated by qRT-PCR, such cytological changes were associated with a significant increase of the pro-inflammatory cytokines and chemokines TNFα, IL1β, and MIP2 expression in BAL cells from the treated group as compared to the control (3.0, 9.7 and 5.6-fold increase, respectively) (Fig. 1). The inflammatory response detected in the bronchoalveolar lavages was confirmed by the microarray data, indeed many GO terms related to inflammation (i.e. immune response, immune cell chemotaxis, inflammatory response, chemokine activity…) were significantly over-represented in the differentially expressed (DE) gene list as compared to the whole microarray (Tables 1 and 2). The expression of

selected genes was confirmed by qRT-PCR and was similar to that observed by microarray for most of the considered genes. A more detailed analysis of the DE genes reveals that inflammatory cytokines (IL6, IL18) and chemokines (CCL2/MCP1, CXCL1/CINC1 and CXCL2/MIP2) were among the most upregulated genes in exposed lungs (Table 5). In addition, among the 363 DE genes, about two dozens were associated with the inflammatory process (Table 5). Moreover, the gene coding for the endothelial cell specific molecule 1 (ESM1), which is involved in the modulation of leukocyte recruitment, but not associated with inflammation-related GO terms, was the most downregulated in bitumen-exposed rats (a 6.9-fold decrease by qPCR) (Table 8). PAH-related detoxification pathways and gene regulation The PAH-inducible cytochrome P450 genes CYP1A1 and CYP1B1 were the most over-expressed in the exposed rat lungs (802.7 and 26.7-fold, respectively) (Table 6). In contrast, the cytochrome P450 gene involved in naphthalene metabolization

Table 4 Cytology of the bronchoalveolar lavages

Control Exposed

Total cells × 103

Macrophages × 103

Neutrophils × 103

Lymphocytes × 103

575.0 ± 278.0 1180.0 ± 275.1⁎

528.2 ± 269.3 (97.6 ± 0.7) 945.0 ± 329.3⁎ (79.4 ± 9.6)⁎

6.1 ± 1.6 (1.3 ± 0.6) 200.3 ± 79.2⁎ (19.3 ± 9.7)⁎

6.4 ± 2.7 (1.4 ± 0.3) 14.7 ± 6.1⁎ (1.3 ± 0.5)

Cell counts were performed using a Malassez hematocytometer and expressed the total cells of the BAL. Cell formula was determined after May-Grunwald Giemsa staining of cytospun cells. Cell percentages are between brackets. ⁎ Statistically different from the control group (P < 0.05).

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Table 5 Differentially expressed genes associated with the inflammatory and immune responses Gene name

Description

Genbank #

CXCL2/ MIP2 SPP1 CXCL1/ CINC1 IL6 CCL2/ MCP1 PPARG

Chemokine (C-X-C motif) ligand 2

NM_053647

Secreted phosphoprotein 1 Chemokine (C-X-C motif) ligand 1

Fold change (qPCR)

GO term

4.7 ± 0.9

6.2 ± 1.2⁎

Inflammatory response; chemotaxis

NM_012881 NM_030845

5.2 ± 0.9 3.8 ± 0.7

4.7 ± 0.8⁎ 3.9 ± 1.0⁎

Inflammatory response; chemotaxis Inflammatory response; chemotaxis

Interleukin 6 Chemokine (C-C motif) ligand 2

NM_012589 NM_031530

4.1 ± 1.8 4.4 ± 2.1

4.6 ± 2.1⁎ 3.9 ± 1.8⁎

Inflammatory response Inflammatory response

NM_013124

1.6 ± 0.2

1.2 ± 0.1

Inflammatory response

NM_017051 NM_019310 NM_053352 NM_053953 NM_013195 NM_022205 M64368 NM_013016

2.2 ± 0.4 2.2 ± 0.2 1.5 ± 0.1 2.3 ± 0.2 −1.5 ± 0.1 −1.7 ± 0.2 1.5 ± 0.1 1.4 ± 0.1

2.4 ± 0.8⁎ 3.4 ± 1.2⁎ 1.3 ± 0.1⁎ 2.4 ± 0.5⁎ −2.1 ± 0.5⁎ −1.9 ± 0.3⁎ n.d. n.d.

Immune response Cytokine binding; chemotaxis Cytokine binding; chemotaxis Cytokine binding Cytokine binding; Immune response Cytokine binding; immune response Chemotaxis; phagocytosis, engulfment Phagocytosis, engulfment

FCGR2B

Peroxisome proliferator activated receptor, gamma Superoxide dismutase 2, mitochondrial Interleukin 8 receptor, alpha Chemokine orphan receptor 1 Interleukin 1 receptor, type II Interleukin 2 receptor, beta chain Chemokine (C-X-C motif) receptor 4 Class III Fc-gamma receptor Protein tyrosine phosphatase, non-receptor type substrate 1 Fc receptor, IgG, low affinity IIb

NM_175756

1.6 ± 0.3

n.d.

FGG CD14 SFLN3 GBP2 C4BPA HP TAP2 VAV1 F3 IL18 CFI

Fibrinogen, gamma polypeptide CD14 antigen Schlafen 3 Guanylate binding protein 2, interferon-inducible Complement component 4 binding protein, alpha Haptoglobin ATP-binding cassette, sub-family B Vav 1 oncogene Coagulation factor 3 Interleukin 18 Complement factor I

NM_012559 NM_021744 NM_053687 NM_133624 NM_012516 NM_012582 NM_032056 NM_012759 NM_013057 NM_019165 NM_024157

1.4 ± 0.1 1.6 ± 0.2 −1.9 ± 0.4 −1.6 ± 0.2 2.4 ± 0.2 1.8 ± 0.2 −2.0 ± 0.4 1.4 ± 0.2 1.6 ± 0.1 2.1 ± 0.2 2.0 ± 0.2

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Inflammatory response; phagocytosis, engulfment Inflammatory response Inflammatory response Immune response Immune response Immune response Immune response Immune response Immune response Immune response Immune response Immune response

SOD2 IL8RA CMKOR1 IL1R2 IL2RB CXCR4 FCGR3 PTPNS1

Fold change (oligo array)

n.d.: not determined. ⁎ Significantly different from the control (Student's t test, P < 0.05).

Table 6 Differentially expressed genes related to PAH metabolization and mechanism of action Gene name

Description

Genbank #

Fold change (oligo array)

Fold change (qPCR)

GO term

CYP1A1

NM_012540

23.2 ± 3.7

2202.6 ± 316.4⁎

NM_012940

22.2 ± 4.0

26.1 ± 4.5⁎

NQO1

Cytochrome P450, family 1, subfamily a, polypeptide 1 Cytochrome P450, family 1, subfamily b, polypeptide 1 NAD(P)H dehydrogenase, quinone 1

NM_017000

4.8 ± 0.3

6.7 ± 0.8⁎

ALDH3A1 GSTA5 SOD2

Aldehyde dehydrogenase family 3, member A1 Glutathione S-transferase A5 Superoxide dismutase 2, mitochondrial

NM_031972 NM_031509 NM_017051

5.6 ± 0.6 2.0 ± 0.2 2.2 ± 0.4

49.8 ± 11.9⁎ 1.9 ± 0.3⁎ 2.4 ± 0.8⁎

HSP1A1 HMOX1

Heat shock 70 kDa protein 1A Heme oxygenase (decycling) 1

NM_031971 NM_012580

2.0 ± 0.2 1.9 ± 0.2

1.7 ± 0.5⁎ 2.0 ± 0.3⁎

XRCC5

X-ray repair complementing defective repair in Chinese hamster cells 5 Glutathione peroxidase 1

NM_177419

1.6 ± 0.1

1.5 ± 0.2⁎

Response to stimulus; aromatic compound metabolism Response to stimulus; aromatic compound metabolism Response to stimulus; xenobiotic metabolism Aldehyde dehydrogenase activity Response to stimulus; response to drug Response to stimulus; response to oxidative stress Response to stimulus; defense response Response to stimulus; DNA damage response Response to stimulus; DNA repair

NM_030826

1.4 ± 0.1

1.3 ± 0.1⁎

Metallothionein Cytochrome P450, family 2, subfamily f, polypeptide 2

NM_138826 NM_019303

1.9 ± 0.2 −2.5 ± 0.4

2.9 ± 0.5⁎ −2.4 ± 0.4⁎

CYP1B1

GPX1 MT1A CYP2F2

⁎ Significantly different from the control (Student's t test, P < 0.05).

Response to stimulus; response to oxidative stress Response to stimulus; metal ion binding Monooxygenase activity

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CYP2F2 had an expression 3.4 times lower in the treated animals. Other inducible genes with an AhR binding site in their promoter including NQO1, ALDH3A1 and GSTA5 were also significantly upregulated in exposed lungs. Moreover, various genes involved in cellular response against oxidative stress including superoxide dismutase 2 (SOD2), heat shock 70 kDa protein 1A (HSP1A1), heme oxygenase 1 (HMOX1), glutathione peroxidase 1 (GPX1) and metallothionein 1a (MT1A) have been found over-expressed in treated animals (Table 6). In addition, the GenMAPP pathway “Rn_Oxidative_stress” was among the few significantly overrepresented pathways (Table 3, Fig. 2). Bitumen induced genes associated with protease activity and inhibition Among the molecular function node, a fairly large number of genes associated with the activity of proteases and their inhibition were found significantly over-represented in the DE gene list (Tables 2 and 7). Other genes and pathways altered by bitumen fumes exposure Monoamine oxidases A and B (MAOA and MAOB) expression was 2.3 and 3.6 times lower in the exposed group, respectively (Table 8).

Moreover, cell cycle regulator gene CDC2A was upregulated 3.3 times following bitumen exposure (Table 8). Discussion The occupational and environmental safety issues represented by bitumen fumes exposure remain to be clarified since experimental and epidemiological studies performed until now do not allow to draw definite conclusions concerning the toxicological effects of this complex mixture. Because of their high contents in carcinogenic PAH, studies concerning the pulmonary mechanisms of action of asphalt have mostly focused on the metabolization and carcinogenic properties of these aromatic compounds. In order to gain additional information, we have undertaken the analysis of the rat lung gene expression profile modifications induced by bitumen fumes following a nose-only exposure. Animals were exposed for 5 consecutive days, 6 h/day to bitumen fumes generated in conditions closed to that observed during road paving operations (Binet et al., 2002a). Lungs were collected immediately after the end of exposure; this time point was believed to be the one which would give us the most information concerning the effects of bitumen fumes since it was assumed that the expression of some genes like those coding for the cytochromes P450 was transient. In addition, it has been shown in a time course experiment that following an

Fig. 2. GenMAPP pathway for oxidative stress. Gray boxes represent genes of which expression was detected in the experiment; stars on the left of some boxes indicate genes of which expression was characterized as statistically modified following bitumen fume exposure; white boxes are used to identify genes of which expression was not detected in the experiment. Numbers on the right of gray boxes indicate the average fold change in gene expression.

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Table 7 Differentially expressed genes associated with protease activity and inhibition Gene name

Description

Genbank #

Fold change (oligo array)

Fold change (qPCR)

GO term

MMP12 ELA1

Matrix metalloproteinase 12 Elastase 1

NM_05396 NM_012552

32.0 ± 0.4 1.7 ± 0.2

n.d. n.d.

HP

Haptoglobin

NM_012582

1.8 ± 0.2

n.d.

SERPINE1

Serine (or cysteine) proteinase inhibitor, class E, member 1 Granzyme A Granzyme K Complement factor I Dipeptidylpeptidase 7

NM_012620

1.9 ± 0.3

n.d.

NM_153468 NM_017119 NM_024157 NM_031973

−1.7 ± 0.3 −1.6 ± 0.2 2.0 ± 0.2 1.7 ± 0.2

n.d. n.d. n.d. n.d.

NM_133617

2.0 ± 0.3

n.d.

AGT

Serine (or cysteine) proteinase inhibitor, clade A member 10 Angiotensinogen

NM_134432

2.0 ± 0.1

n.d.

CSTB ITIH4

Cystatin B Alpha-trypsin inhibitor, heavy chain 4

NM_012838 NM_019369

1.6 ± 0.2 2.6 ± 0.3

n.d. n.d.

PTTG1 FETUB

Pituitary tumor-transforming 1 Fetuin beta

NM_022391 NM_053348

1.7 ± 0.2 2.1 ± 0.2

n.d. n.d.

Elastase activity Elastase activity; serine-type endopeptidase activity Serine-type endopeptidase inhibitor activity Serine-type endopeptidase inhibitor activity Serine-type endopeptidase activity Serine-type endopeptidase activity Serine-type endopeptidase activity Serine-type endopeptidase inhibitor activity Serine-type endopeptidase inhibitor activity Serine-type endopeptidase inhibitor activity Cysteine protease inhibitor activity Serine-type endopeptidase inhibitor activity Cysteine protease inhibitor activity Cysteine protease inhibitor activity

GZMA GZMK CFI DPP7 SERPINA10

n.d.: not determined.

intratracheal instillation of urban particulate matter, the largest variations in gene expression were observed shortly (2–6 h) after animal exposure (Kooter et al., 2005). Then rat lung gene expression profile alterations detected immediately after the end of animal exposure can be considered as an approximate of that would be observed in workers. Biostatistical analyses of the raw data generated from microarray experiments yielded a list of 363 genes and EST differentially expressed following bitumen exposure. In combination to this global gene expression profiling approach, we used the Gene Ontology tools and a pathway analysis software for identifying physiological processes altered by asphalt fumes. The most interesting finding was that bitumen fumes induced a pulmonary inflammatory response which was detected by BAL analysis and microarray. This was a fairly new result since among the few experimental studies concerning rat inhalation of bitumen fumes, none reported any pulmonary inflammation response as a consequence of bitumen exposure (Ma et al., 2003; Bottin et al., in press). Our finding can be explained by the possible transient inflammatory response induced following exposure; indeed once the pulmonary clearance of particles has been achieved, inflammation tends to disappear. Since we

performed the analysis immediately after the end of exposure, particle clearance was occurring and an inflammatory response was observable. In contrast, in the Bottin et al.'s (in press) experiment, although BigBlue® transgenic rats were exposed to a similar aerosol, the inflammatory response was analyzed 3 days after the end of bitumen fume inhalation, by this time the particle clearance might have been completed. Similar conclusions could be drawn from the Ma et al.'s (2003) study during which analyses were performed the day after the end of exposure. In addition, they used a bitumen fume generator and a rat strain different from that used in our study and the TPM concentrations were at most half of that we generated in our work, this may also explain in part the differences between their results and ours. However, one study showed the presence of nasal epithelium inflammation in rats following bitumen fume inhalation (Sikora et al., 2003). Nonetheless, our results are in agreement with the fact that generated bitumen fumes contained particles (mass median aerodynamic diameter 2.6 ± 0.1 μm) which had to be cleared by alveolar macrophages. In addition, the pulmonary inflammatory response in rats following inhalation of bitumen fumes can be compared to that observed with other aerosols such as diesel exhausts, which also contain particles and PAH (Dybdahl et al., 2004; Rao et al., 2005).

Table 8 Miscellaneous differentially expressed genes of interest Gene name

Description

Genbank #

ESM1 Endothelial cell-specific molecule 1 NM_022604 MAOA Monoamine oxidase A XM_343764 MAOB Monoamine oxidase B NM_013198 CDC2A Cell division cycle 2 homolog A (S. pombe) NM_019296 ⁎ Significantly different from the control (Student's t test, P < 0.05).

Fold change (oligo array)

Fold change (qPCR)

GO term

−4.7 ± 1.0 −1.7 ± 0.3 −2.1 ± 0.2 3.3 ± 0.3

−4.9 ± 1.2⁎ −2.2 ± 0.4⁎ −4.4 ± 1.4⁎ 2.4 ± 0.5*

Extracellular space Amine oxidase activity Amine oxidase activity Mitotic G2 checkpoint

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As observed in this study, asphalt fumes induced an overexpression of TNFα by alveolar macrophages, this cytokine plays a key role in lung inflammation. Indeed, it triggers the expression of pro-inflammatory chemokines and cytokines such as MIP2 (CXCL2), CINC1 (CXCL1) or IL6 by lung epithelial cells; these molecules then contribute to the recruitment and activation of inflammatory cells (macrophages and granulocytes) (Driscoll et al., 1997; Driscoll, 2000). Our results are in agreement with the literature since the inflammatory response that we observed following bitumen exposure was associated with a significant upregulation of many pro-inflammatory chemokines. In addition, the in vitro cytokine-regulated gene ESM1, which codes for an endothelial specific molecule (Lassalle et al., 1996) involved in the modulation of recruitment of circulating leukocytes (Bechard et al., 2001), is underexpressed in bitumen-exposed rats. Such data represent another evidence that bitumen may induce an inflammatory response by triggering the recruitment of circulating white blood cells. During inflammation, macrophages produce matrix metalloprotease 12 (Churg and Wright, 2005), an enzyme tightly linked with emphysema (Churg and Wright, 2005). This protease is also synthesized by lung epithelial cells and its expression is induced by TNFα (Lavigne et al., 2004; Lavigne and Eppihimer, 2005). Then, the over-expression of this enzyme in bitumen-exposed lungs is consistent with the inflammatory response observed in treated animals. In addition, various serine-protease (i.e. elastase) inhibitors are over-expressed in exposed rats; these molecules are required for the protection of the lung against the adverse effects of proteases. Indeed, the balance between proteases and their inhibitors is required for lung homeostasis, thus any modifications of this equilibrium toward an accumulation of proteases is a prerequisite to emphysema and subsequently chronic obstructive pulmonary deficiency (COPD) (Spurzem and Rennard, 2005). Since epidemiological studies showed that lung function decline pathologies such as COPD were significantly more prevalent among asphalt workers (Randem et al., 2004), it could be assumed that chronic exposure to bitumen fumes may induce a shift in the protease/antiprotease balance toward proteases which would be responsible for such non-malignant pulmonary diseases as previously shown in smokers (Churg and Wright, 2005). Alveolar recruitment of neutrophilic granulocytes and macrophages contributes to the production of deleterious reactive oxygen species (ROS) and especially nanomolar concentrations of superoxide anion O2 − (Nagata, 2005). As a consequence of such toxic insult, cellular antioxidant mechanisms are mobilized; it has been shown that detoxifying enzymes such as manganese-dependent superxodide dismutase (MnSOD or SOD2), and glutathione peroxidase (GPX) were overexpressed following silica particle-induced inflammation (Janssen et al., 1992). Similarly, we observed an upregulation of these two enzymes following bitumen fumes exposure. Other antioxidant genes such as MT1A, HMOX1 and GSTA5 induced by oxidative stress were also over-expressed following bitumen exposure; their expression may be stimulated by the presence of an antioxidant response element (ARE) in their promoter •

(Primiano et al., 1997). Oxidative stress generated during inflammation may cause cellular damages but also stimulate the proliferation of type II alveolar epithelial cells as shown in an experimental study using hydrogen peroxide (Sigaud et al., 2005). Proliferation of such cells is associated with the overexpression of cell cycle regulating protein such as CDC2A as previously shown (Wu et al., 1995). These experimental data corroborate our results showing an increase in RNA levels of CDC2A suggesting a stimulation of the cell proliferation following bitumen exposure as a possible consequence of the damaged cells replacement. Interestingly, no relevant expression modification of apoptosis-related genes was detected despite the probable presence of toxic insult associated with oxidative stress. This may be due to the fact that either the oxidative stress induced by bitumen fumes was not sufficient to trigger any detectable apoptosis, or during programmed cell death many proteins involved in this process undergo posttranslational modifications without changes in their expression (Haddad, 2004). Oxidative stress can also be induced by free and particlebond PAH in which metabolization leads to the generation of superoxide anion during quinone metabolites formation (Miller and Ramos, 2001). Furthermore, PAH activate the transcription of many genes because of their binding to the nuclear receptor Aromatic hydrocarbon Receptor (AhR) which can bind to specific sites in the promoter of various genes such as CYP1A1, CYP1B1, NQO1, ALDH3 and GSTA5 (Hankinson, 1995). Such genes are induced in our experiment following bitumen fume exposure which is corroborated by previously published papers showing that inhalation of bitumen fumes leads to the activation of metabolic pathways involving these genes in rat lung (Ma et al., 2002, 2003). In contrast, another phase I metabolism enzyme CYP2F2, is downregulated in bitumen-exposed lungs. This cytochrome P450 is involved in the metabolic activation of naphthalene, into a toxic metabolite (Shultz et al., 1999). Since high amounts of naphthalene are found in bitumen fumes (Micillino et al., 2002), CYP2F2 decreased expression might be related to a pulmonary defense mechanism responsible for the lack of rat lung cytotoxicity from this PAH (Baldwin et al., 2005). Inhalation of bitumen fumes induced the formation of DNA adducts in lungs (Genevois-Charmeau et al., 2001; Binet et al., 2002b; Wang et al., 2003) which are a consequence of polycyclic aromatic hydrocarbon transformation into PAHdihydrodiol-epoxides by CYP1A1 and CYP1B1 (Miller and Ramos, 2001). Such DNA adductions are responsible for DNA strand break and mutation; various enzymes have been associated with DNA adduct removal and DNA repair. However, no gene involved in DNA damage detection or repair with the exception of XRCC5 was upregulated in treated animals. Such observations might be associated with either the constitutive expression of such genes or the fact that the extent of DNA damages induced by PAH might not be sufficient to detectably trigger their expression. In addition, monoamine oxidases A and B which are involved in the metabolism of xenobiotics (Strolin Benedetti and Tipton, 1998) and neurotransmitters (Shih et al., 1999),

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were downregulated in response to bitumen fume exposure. Considering that monoamine oxidase expression deficiency (Burnet et al., 2001) is associated with alterations of pulmonary functions, the repressed expression of these enzymes could be associated either with the regulation of the animal breathing process in response to fume exposure or the prevention of metabolization of xenobiotics into toxic metabolites. Altogether, this first study by an oligonucleotide microarray analysis approach shows that the exposure to bitumen fumes leads to the activation of biological processes involved in inflammatory response, xenobiotic metabolization and antioxidant defense. Such alterations induced by bitumen fumes could be related to their PAH and particle content. In addition, these data may represent a starting point for the explanation of results obtained during epidemiological studies regarding the respiratory airway diseases observed in bitumen workers. Then, this approach contributes to validate our experimental design and appears to be a valuable tool for assessing the occupational health risk associated with the exposure to asphalt fumes. Such type of study may also allow to better predict the biological effects of chemicals and may help in the development of better occupational or environmental protection systems. Acknowledgments The authors gratefully thank Drs D. Goidin and C. Thissot for their technical support. References Baldwin, R.M., Shultz, M.A., Buckpitt, A.R., 2005. Bioactivation of the pulmonary toxicants naphthalene and 1-nitronaphthalene by rat CYP2F4. J. Pharmacol. Exp. Ther. 312, 857–865. Bechard, D., Scherpereel, A., Hammad, H., Gentina, T., Tsicopoulos, A., Aumercier, M., Pestel, J., Dessaint, J.P., Tonnel, A.B., Lassalle, P., 2001. Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1. J. Immunol. 167, 3099–3106. Beissbarth, T., Speed, T.P., 2004. GOstat: find statistically overrepresented gene ontologies within a group of genes. Bioinformatics 20, 1464–1465. Binet, S., Bonnet, P., Brandt, H., Castegnaro, M., Delsaut, P., Fabries, J.F., Huynh, C.K., Lafontaine, M., Morel, G., Nunge, H., Rihn, B., Vu Duc, T., Wrobel, R., 2002a. Development and validation of a new bitumen fume generation system which generates polycyclic aromatic hydrocarbon concentrations proportional to fume concentrations. Ann. Occup. Hyg. 46, 617–628. Binet, S., Pfohl-Leszkowicz, A., Brandt, H., Lafontaine, M., Castegnaro, M., 2002b. Bitumen fumes: review of work on the potential risk to workers and the present knowledge on its origin. Sci. Total Environ. 300, 37–49. Bottin, M.C., Gate, L., Rihn, B.H., Micillino, J.C., Monhoven, N., Nunge, H., Morel, G., Wrobel, R., Ayi-Fanou, L., Champmartin, C., Keith, G., Binet, S., in press. Genotoxic effect of bitumen fumes in Big Blue transgenic rat lung. Mutat. Res. Burczynski, M.E., McMillian, M., Ciervo, J., Li, L., Parker, J.B., Dunn II, R.T., Hicken, S., Farr, S., Johnson, M.D., 2000. Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells. Toxicol. Sci. 58, 399–415. Burgaz, S., Erdem, O., Karahalil, B., Karakaya, A.E., 1998. Cytogenetic biomonitoring of workers exposed to bitumen fumes. Mutat. Res. 419, 123–130. Burnet, H., Bevengut, M., Chakri, F., Bou-Flores, C., Coulon, P., Gaytan, S.,

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