Particle Toxicology - Chapter 11 pot

As shown inFigure 11.1a, DEP and their organic extract induce a transient CYP1A1 mRNA expression in human bronchial epithelial cells HBE similar to BaP whereas carbon black particles hav

11 Particle-Associated Organics

and Proinflammatory Signaling

Francelyne Marano, Sonja Boland, and Armelle

Baeza-Squiban

Laboratoire de Cytophysiologie et Toxicologie Cellulaire,

Universite´ Paris 7 – Denis Dide`rot

CONTENTS

11.1 Introduction 211

11.2 What Is the Role of These Organic Compounds in the Effects of PM? 211

11.2.1 The Particles Organic Fraction 212

11.2.2 Bioavailability of Organic Compounds 213

11.2.3 Organic Compounds and Oxidative Stress 214

11.2.4 Organic Compounds and Inflammation 216

11.3 Conclusion 221

Abbreviations 221

References 221

11.1 INTRODUCTION

The burning of fossil fuels generates fine and ultrafine airborne particles, which contain a large amount of organic compounds including polyaromatic hydrocarbons (PAH) These particles must

be taken into account as they are considered to be among the most abundant components of particulate matter 2.5 mm (PM2.5) in urban areas Most of them are produced by diesel engine-powered cars and diesel exhaust particles (DEP) constitute, on average, 40% of the PM10in a city such as Los Angeles (Diaz-Sanchez 1997), and in a kerbside station in Paris more than 50% of particles were close to the ultrafine range (%0.26 mm), likely due to the influence of the traffic (Baulig et al 2004) Chemical analysis between PM2.5collected in a kerbside and a background station in Paris revealed that PAH are twice as important in the kerbside station The results are more relevant with heavy PAH than light PAH, due to their higher stability We have also observed variations of PAH according to the seasons, probably due to chemical reactions with atmospheric oxidants However, PAH are only a part of the organic component and they do not greatly influence the soluble organic fraction (SOF) measured after dichloromethane extraction that appear to be between 10 and 12% of the mass of the particles whatever the station and approximately 45% lower than the SOF of DEP (20%) (Baulig et al 2004)

11.2 WHAT IS THE ROLE OF THESE ORGANIC COMPOUNDS

IN THE EFFECTS OF PM?

In this chapter, we do not consider the genotoxic and carcinogenic effects of these organic compounds The dramatic increase of human allergic airway diseases in the last century has followed

211

the increase in the use of fossil fuels and many epidemiological studies have provided indirect evidence for a correlation between particulate pollution and increased incidence of asthma and allergic rhinitis Numerous experimental studies in animals, in human volunteers, and in vitro were performed to provide a causal explanation for these observations Diaz-Sanchez et al have published numerous studies on the role of DEP and their associated PAH in the induction of allergic airway diseases (Riedl and Diaz-Sanchez 2005) In vivo nasal provocation studies, using amounts of DEP equivalent to the total exposure in 1–3 days in Los Angeles, showed enhanced immunoglobulin

E (IgE) production in the human upper airway This response appears to be linked to organic compounds of DEP since it has been established that PAH–DEP could significantly increase IgE mRNA and protein production in IgE-secreting Epstein–Barr-virus transformed human B cells

in vitro (Tsien et al 1997) The effects of DEP were specific since they do not increase IgG, IgA,

or IgM (Diaz-Sanchez et al 1994) The ability of DEP to act as an adjuvant was tested by performing nasal provocation challenges with DEP, ragweed antigen Amb a 1, or both simultaneously in ragweed-sensitive subjects (Diaz-Sanchez 1997) Ragweed-specific IgE was 16 times higher following ragweed plus DEP challenge compared with ragweed alone However, IgG levels remained constant after challenge with DEP plus antigen Further experiments have shown an increase in cytokine mRNA levels such as Interleukin-2 (IL-2), IL-4, IL-5, IL-6, and Interferon g The susceptibility to the adjuvant effect of DEP is an intrinsic trait, as there is a high intraindividual reproducibility of the nasal allergic responses in human exposure studies (Bastain et al 2003) Several candidate genes could be involved in this susceptibility to particles, such as antioxidant enzymes or Toll Like Receptor 4, CD14 or Tumor Necrosis Factor a (reviewed in Granum and Lovik 2002) This adjuvant effect of DEP for allergic sensitization is a delayed response and does not explain acute PM effects on airway hyperreactivity It has been recently demonstrated by Hao (Hao

et al 2003), using BALB/c mouse model sensitized by ovalbumin, that aerosolized DEP could induce increased airway hyperreactivity even if DEP delivery is delayed after the peak inflammatory response It was concluded that DEP induced airway hyperreactivity independently of adjuvant effects Interestingly, DEP co-administration with a neoallergen such as keyhole limpet hemocyanin shows that the particles could synergize with the neoallergen and drive the de novo production of antigen-specific IgE (Diaz-Sanchez et al 1999) These results suggest that DEP exposure with a neoallergen leads to sensitization IgE mucosal production Thus particulate air pollution may influ-ence both—the sensitization and the provocation phase of allergy by inducing oxidative and inflammatory reactions in the respiratory mucosa (Granum and Lovik 2002)

Moreover, various animal experiments suggest that DEP may alter both innate and acquired cellular immunity Beside their adjuvant effect, these particles have also an immunosuppressive effect in animals The increased susceptibility of the lung to infection in rats exposed to DEP was related to the inhibition of the functions of alveolar macrophages by organic compounds, but not the carbonaceous core (Castranova et al 2001) Using nitric oxide (NO) production as a marker of macrophage function, it was shown that crude DEP organic extracts inhibit both Lipopolysac-charide and Bacillus Calmette–Gue´rin (BCG) induced NO production by a murine macrophage cell line explaining the impaired bacterial clearance noticed in a BCG mouse lung infection model (Saxena et al 2003b) By fractionation of the organic extract, it appears that this inhibitory effect was mainly due to PAH and resin fractions (Saxena et al 2003a)

The identification of the chemical components involved in these biological effects and the understanding of the underlying mechanisms are still imperfect Such studies are difficult, as there exists a great variability in the chemical composition of PM according to their emission sources, age, and site of sampling

The organic fraction of particles comprises a countless quantity of compounds (such as aliphatic hydrocarbons, PAH, nitroaromatics hydrocarbons, quinones, aldehydes, and heterocyclics), some

of which are still unidentified This fraction can represent up to 50% of the mass of the particle and may contain toxic compounds At the present time, PAH are quantitatively and qualitatively the best known family of organic compounds adsorbed on particles, although they only represent a few percent of the organic fraction The interest in these compounds lies in their known genotoxic and inflammatory properties and their use as a tracer of source They have been shown to be in higher concentrations in submicron particles (De Kok et al 2005; Rehwagen et al 2005), which can be explained by the fact that soot from combustion sources consist primarily of fine particles with high PAH content and that the smaller particles have a relatively high surface area for PAH adsorption (Ravindra, Mittal, and Van Grieken 2001)

Another category of organic compounds that has held the attention of biologists are quinones, due to their ability to induce various hazardous effects in vivo such as acute cytotoxicity, immu-notoxicity, and carcinogenesis (Bolton et al 2000) Four quinones (1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-phenanthraquinone, 9,10-anthraquinone) have been identified and quan-tified in DEP (7.9–4.04 mg/g) and in Los Angeles PM2.5(5–730 pg/m3) (Cho et al 2004)

The presence on particles of organic compounds exhibiting a potential biological effect raises the question of their bioavailablity To understand the processes whereby particles deliver and transfer toxic components to target cells, experiments have been done using radio-labeled benzo(a)pyrene (B(a)P)-bound denuded particles After their administration to dogs, the extent and rate of release as well as their metabolic fate were investigated (Gerde et al 2001b) It reveals that in the alveolar region, B(a)P was adsorbed mostly unaltered into the blood and was systematically metabolized (Gerde et al 2001a) In the conducting airways,

a smaller fraction of B(a)P was slowly deposited but metabolized in the airway epithelium (Gerde et al 2001b) Nevertheless, a large fraction of B(a)P remained bound to particles even

6 months after the exposure (Gerde et al 2001b)

In cells, foreign substances are detoxified by two sequential reaction processes, namely, Phase I and Phase II In Phase I reactions, xenobiotics are mainly oxidized by cytochrome P450 (CYP) enzymes to become more polarized metabolites Phase II metabolism, catalyzed by enzymes such

as glutathione S-transferase (GST) and NADP(P)H:quinone oxidoreductase (NQO1), converts the reactive Phase I metabolites to more hydrophilic substances, allowing their elimination

Among the members of the CYP gene family, CYP1 is known to be induced by PAH through a receptor-dependent mechanism The cytosolic aryl hydrocarbon receptor (AhR), when bound by PAH, translocates to the nucleus, heterodimerizes with another partner, and activates the transcrip-tion of CYP1 family genes through binding to the xenobiotic response element Native DEP, PM and their respective extracts act as activators of the AhR, inducing CYP1A1 expression and activity (Meek 1998; Bonvallot et al 2001; Baulig et al 2003a) As shown inFigure 11.1a, DEP and their organic extract induce a transient CYP1A1 mRNA expression in human bronchial epithelial cells (HBE) similar to B(a)P whereas carbon black particles have not such an effect (Baulig et al 2003a) The genes of Phase II metabolism (GST, NQO-1) are regulated in a concerted manner at the transcriptional level through the antioxidant-responsive element (ARE)/electrophile-responsive element The transcription factor NF-E2-related factor-2 (Nrf2) is central to ARE-mediated gene expression (Itoh et al 1997) and Nrf2 (K/K) mice exhibit significant reduction of phase II enzymes (Cho et al 2002) DEP induce the translocation of Nrf2 to the nucleus of HBE cells, increase nuclear protein binding to the ARE (Baulig et al 2003a), as well as NQO1 expression as shown in Figure 11.1b

Whereas this biotransformation process aimed to detoxify xenobiotics, a bioactivation may occur and reactive metabolites are produced especially during the Phase I By this way, PAH give rise to electrophilic metabolites responsible for their genotoxicity

The observation of the CYP1A1 gene induction in lung homogenates of Big Blue rats exposed, by inhalation, to whole DEP fumes supports the argument that the leaching of organic compounds from particles can occur (Sato et al 2000) Moreover, in another study,

it was shown that this transient CYP1A1 induction in lungs of rats only occurs with DEP and not with carbon black (Ma and Ma 2002) The mechanisms of transfer of organic compounds from particles to the target cells can involve the uptake of particles (Bonvallot et al 2001) However, a recent study using fresh butadiene soots suggests that the transfer to cells occurs

by the direct contact between soots and the plasma membrane, likely involving a partitioning mechanism (Penn et al 2005)

Moreover, it is not known how effectively biological media (e.g., serum or interstitial fluids) can solubilize the organic compounds (Keane et al 1991) It has been shown that the addition of surfactant in an aqueous suspension of DEP or carbon black particles on which a PAH mixture has been previously adsorbed doesn’t favor the leaching of PAH (Borm et al 2005) In addition, the PAH bioavailability is negligible when the PAH content is low relative to the particle monolayer surface (Borm et al 2005)

Evidence for the involvement of oxidative stress in the effects of organic compounds came from the initial observation that the mortality resulting from lung edema after intratracheal

2.7

1.7

NQO1

CYP1A1

18S

2 h

(a)

(b)

FIGURE 11.1 Induction of cytochrome P-450 1A1 (CYP1A1) and NADPH: quinone oxidoreductase 1 (NQO-1) gene expression in HBE cells (a and b respectively) Cells were treated or not with DEP (10 mg/cm2), carbon black (10 mg/cm2) organic extracts of DEP (OE-DEP, 10 mg/mL) or benzo(a)pyrene (B(a)P, 3 mM) RNA (30 mg) were extracted from cells after 2, 6, 24, or 48 h of treatment, electrophoresed, Northern-blotted, and then incubated with a32P-labeled cDNA probe for CYP1A mRNA, NQO-1 mRNA, or 18S RNA (From Baulig, A., Garlatti, M., Bonvallot, V., Marchand, A., Barouki, R., Marano, F., and Baeza-Squiban, A., Am J Physiol Lung Cell Mol Physiol., 285, L671–L679, 2003a With permission)

administration of whole DEP into mice was suppressed by pretreatment with polyethylene glycol-modified superoxide dismutase (Sagai et al 1993) and that it was limited with methanol-washed DEP In this same study, it was shown that in acellular conditions, whole DEP produced oxygen radicals (superoxide anion radical O$K

2 and hydroxyl radical%OH) ident-ified by electron paramagnetic resonance, which were not produced with methanol-washed DEP (Sagai et al 1993) Quinones have been reported to be responsible for this radical production due to their ability to undergo enzymatic (P450/P450 reductase) and non enzymatic redox cycling with their corresponding semiquinone radical giving rise to O$K

2 (Bolton et al 2000) Further enzymatic or spontaneous dismutation of O$K

2 produces hydrogen peroxide, which in presence of trace amounts of transition metals such as iron gives%OH by the Fenton reaction A methanol extract of DEP has been shown to cause a significant formation of O$K

2 in the presence

of Cyt P450 reductase (Kumagai et al 1997), an enzyme which activity is increased in DEP-treated mice (Lim et al 1998) More recently, PM2.5have been found to contain abundant and stable semiquinone radicals detected by EPR and to induce DNA damage (Dellinger et al 2001; Squadrito et al 2001) To these semiquinone radicals directly present on particles, others can be produced during an alternative PAH-metabolization involving dihydrodiol dehydrogenase leading to the generation of PAH o-quinones (Penning et al 1999) Furthermore, the CYP1A1 catalytic activity generates reactive oxygen species (ROS) (Perret and Pompon 1998) Redox-active transition metals, redox cycling quinones, and PAH present on PM can act synergistically to produce ROS

Taken altogether, these data reveal that organic compounds are a source of ROS It explains the pro-oxidant status measured using various specific fluorescent probes in airway epithelial cells and macrophages treated either with DEP, PM, or their corresponding organic extract, whereas carbon black particles or solvent-extracted particles do not have such an effect (Hiura

et al 1999; Li et al 2002; Baulig et al 2003a; Baulig et al 2004) For example, increased ROS production determined by the dichlorofluorescein fluorescence was observed in HBE cells exposed for 4 h to DEP, urban PM2.5sampled in Paris, and their respective extracts The extracts gave a fluorescence signal similar to native particles (Figure 11.2) A pro-oxidant status is known

to induce cellular specific responses in the order that cells face oxidant insult Various studies have shown that such responses occur in DEP-treated cells By a genomic approach, the expression profiles of genes induced by organic extracts of DEP in rat alveolar macrophages reveals the increased expression of anti-oxidant enzymes (heme oxygenase (HO-1), thioredoxin peroxidase 2, NADPH dehydrogenase) (Koike et al 2002; Koike et al 2004) Similarly, the overexpression of HO-1 was observed in a murine macrophages cell line (RAW264.7) exposed

to organic extracts of DEP as well as in epithelial cells (Li et al 2002) These data were completed by observation of a change in the proteome of RAW264.7 exposed to DEP organic extracts 51 proteins were newly expressed but were suppressed by N-acetylcysteine, a thiol antioxidant (Xiao et al 2003)

Furthermore, from crude DEP extracts, Li and collaborators (Li et al 2000) have shown that only the polar fraction that is enriched in quinones and the aromatic fraction enriched in PAH were able to decrease the cellular GSH/GSSG ratio in macrophages as well as to induce HO-1 expression, both indicative of a situation of oxidative stress In other respects, the comparison of coarse, fine, and ultrafine PM revealed that ultrafine PM have the highest redox activity (Cho et al 2005), in agreement with the observation that ultrafines were the most potent towards inducing HO-1 expression and depleting intracellular GSH (Li et al 2003) However, the susceptibility to an adverse health effect of DEP is linked to the functional variation in natural antioxidant defenses The polymorphism of GST genes has been associated with atopy and experimental studies provide evidence that the GSTM1 and GSTP1 genotypes can play a role in the susceptibility to the adjuvant effect of DEP (Gilliland

et al 2004)

11.2.4 ORGANIC COMPOUNDS ANDINFLAMMATION

In vivo human exposure studies show that phenantrene, in contrast to carbon black, increase IgE production, but did not cause inflammatory cell infiltration (Saxon and Diaz-Sanchez 2000)

In animal studies however PAH not only increased IgE production (Heo, Saxon, and Hankinson 2001) but also the recruitment of inflammatory cells (Hiyoshi et al 2005) Furthermore, in vitro studies have shown that organic compounds are involved in the proinflammatory response induced

by particles in the two respiratory target cells (airway epithelial cells and macrophages) Several studies using HBE cell lines (BEAS-2B, 16HBE), normal human airway epithelial cells, and macro-phages have shown that an inflammatory mediator release (IL-8, GM-CSF, RANTES, TNF-a) can

be induced by exposure to DEP extract (Boland et al 2000; Fahy et al 2000; Li et al 2002; Vogel

et al 2005) From the comparison of native DEP with their organic extracts obtained with benzene (Kawasaki et al 2001) or dichloromethane extraction with extracted DEP and carbon black particles (Figure 11.3a) (Boland et al 2000), it was concluded that organic compounds mimic native DEP and that the carbonaceous core is not involved in the proinflammatory response Moreover, the role of organic compounds was strengthened by the observation that DEP from vehicles equipped with a catalytic converter exhibiting a SOF of 8.3% induce a lower GM-CSF release by HBE cells than DEP from non-equipped vehicles having a 35% SOF (Figure 11.3b) (Boland et al 2000) Concerning PM, until now, few studies have addressed the involvement of organic compounds

By chemical fractionation (organic vs aqueous fraction) of Paris urban PM2.5, it was shown that the GM-CSF secretion induced by native PM2.5in HBE cells was mimicked by their organic extracts

0

50

100

150

200

250

300

350

400

450

FIGURE 11.2 Dichlorofluorescein (DCF) fluorescence intensity in human bronchial epithelial cells treated with diesel exhaust particles (DEP, 10 mg/cm2) or their corresponding organic extract (OE-DEP), Paris urban

PM2.5 (PM, 10 mg/cm2) or their corresponding organic extract (OE-PM), or carbon black particles (CB,

10 mg/cm2) The cells were loaded with 20,70-dichlorofluorescein-diacetate (H2DCF-DA) at 20 mM for

20 min and then treated or not with the toxics for 4 h The DCF fluorescence was measured by cytometry Results are expressed in % of increase of DCF fluorescence relative to control

whereas the aqueous extract had a slight effect (Baeza-Squiban et al 2005) The absence of effect of the soluble fraction was also observed with PM10(EHC-93) in normal HBE cells (Fujii et al 2001) The effect of urban particles on the cytokine production of macrophages was also due to the organic fraction (Vogel et al 2005) (Table 11.1)

The induction of chemokine release may be responsible for the inflammatory cell infiltration observed in the in vivo studies (Hiyoshi et al 2005) Figure 11.4 shows the inflammatory response, which may result from leukocyte recruitment and activation Beside the stimulation

of these inflammatory cells by locally secreted cytokines, it has also been shown that PAH have direct effects on leucocytes Pyrene increase the production of IL-4 by T lymphocytes (Bommel

et al 2000) and organic extracts of DEP increase CD1a and costimulatory molecule expression on monocyte derived dendritic cells (Koike and Kobayashi 2005), IgE production of B lymphocytes (Takenaka et al 1995; Tsien et al 1997), IL-4 and histamine release from basophils (Devouassoux et al 2002) as well as mast cell (Diaz-Sanchez, Penichet-Garcia, and Saxon 2000) and eosinophil degranulation (Terada et al 1997) This release of granulocyte mediators

100

80

60

40

20

0

C (a)

(b)

DEP Carbonblack

- cata OE-DEP- cata + cataDEP OE-DEP+ cata

100

120

140

80

60

40

20

0

FIGURE 11.3 GM-CSF release by HBE cells treated for 24 h with (a) DEP, extracted DEP, or carbon black particles at 10 mg/cm2and dichloromethane extracts of DEP (OE-DEP) at 20 mg/mL, (b) DEP collected from

a diesel engine with and without an oxidation catalyst and their corresponding organic extracts (OE-DEP)

*P!0.05 compared with control value, BP!0.05 compared with DEP-treated culture (From Boland, S., Bonvallot, V., Fournier, T., Baeza-Squiban, A., Aubier, M., and Marano, F., Am J Physiol Lung Cell Mol Physiol 278, L25–L32, 2000 With permission)

TABLE 11.1

Inflammatory Effects of Organic Compounds

In vivo studies in humans

CB increase the number of inflammatory cells but NOT IgE Saxon and

Diaz-Sanchez (2000) Phenanthrene (with or without allergen) increase IgE but NOT

inflammatory cells

In vivo studies in animals

Mice Phenanthraquinone increase neutrophils, eosinophils, IL-5 and

CB and organic extracts of DEP enhance ovalbumin specific IgE and

Organic extracts of DEP increase neutrophil number in response to LPS but did NOT effect LPS induced cytokine secretion Yanagisawa et al.(2003) PAH enhance IgE production in response to allergen Kanoh et al (1996)

In vitro studies

Epithelial cells Organic extracts of DEP increase histamine receptor in nasal

epithelial cells as well as histamine-induced IL-8 and GM-CSF release

Terada et al (1999)

Organic extracts of DEP increase GM-CSF Boland et al (1999) DEP but not CB stimulate amphiregulin secretion Blanchet et al (2004) Organic extracts of DEP increase IL-8 and HO-1 Li et al (2002) Pyrene increase IL-8 but NOT eotaxin expression in A549 cells Bommel et al (2003) B(a)P and organic extracts of DEP increase IL-8, GM-CSF and

Alveolar macrophages Organic extracts of DEP and PM increase IL-8, TNF and COX2 Vogel et al (2005)

Organic extracts of DEP increase IL-8 Li et al (2002) Organic extracts of DEP did NOT alter costimulatory molecules (B7)

and MHC class II (CDIa) expression and antigen presentation

Koike and Kobayashi (2005)

Organic extracts of DEP decrease PGE2 production by blocking COX-2 enzyme activity

Rudra-Ganguly et al (2002)

Organic extracts of DEP reduce production of IL-1and TNF-a in response to inflammatory agents Siegel et al (2004) Organic extracts of DEP increase IL-1 but not TNF-a Yang et al (1997) Peripheral blood

mononuclear

cells (PBMC)

Organic extracts of DEP and PAH in presence of LPS increase or decrease IL-10 production depending on the order of exposure

Pacheco et al (2001)

Organic extracts of DEP decrease MCP-1 but increase IL-8, RANTES and chemotactic activity for neutrophils and eosinophils Fahy et al (1999) Organic extracts of DEP decrease IP10 and increase MDC production

Organic extracts of DEP increase IL-8, RANRES and TNFa in PBMC of allergic persons

Fahy et al (2000) MonoDC Organic extracts of DEP increase the expression of costimulatory

molecules (B7) and MHC class II (CDIa) expression and enhance allergen presentation

Koike and Kobayashi (2005)

PAH during monoDC differentiation decrease expression of CD1a, B7.1 and CD40 as well as DC function Laupeze et al (2002) Lymph node cells B(a)P induce IL-4 and IL-6 production Fujimaki et al (1997)

T lymphocyte Pyrene increases IL-4 production Bommel et al (2000)

(continued)

Table 11.1 (Continued)

B lymphocyte Organic extracts of DEP increase IgE production Tsien et al (1997)

Organic extracts of DEP increase IgE production only in the presence

Basophils Organic extracts of DEP increase IL-4 and histamine release in cells

from allergic and non-allergic subjects Devouassoux et al.(2002) PAH increase IL-4 and histamine release only in presence of IgE Kepley et al (2003) Mast cell Organic extracts of DEP increase histamine release in the presence of

IgE

Diaz-Sanchez et al (2000) Eosinophils Organic extracts of DEP increase degranulation and adhesion to

epithelial cells

Terada et al (1997)

BP IgE

Histamine tryptase Lipid mediators Cytokines Cytokines

(IL-1, IL-4, IL-5, IL-6,

IL-10, GM-CSF, TNFα)

Chemokines

(IL-8, MCP, Eotaxin,

MC

Epithelium

NP

MBP, ECP, EPO Lipid mediators Cytokines

Chronic asthma Epithelial damage bronchial remodeling chronic inflammation bronchial

hyperresponsiveness smooth muscle contraction

Acute asthma Mucus secretion vasodilatation and vasopermeation bronchial hyperresponsiveness smooth muscle contraction APC

Chemotaxis and transmigration of Leucocytes

Adhesion molecules endothelium + Ag

PAH

Adhesion molecules

+ Ag Histamine receptor

AM

MPO Proteinases Lipid mediators

CD1a B7

IL-5 GM-CSF

IL-4 IL-6

FIGURE 11.4 Scheme of the inflammatory response induced by PAH PAH stimulate alveolar macrophages (AM) and epithelial cells to release cytokines and chemokines, inducing the recruitment of peripheral blood leukocytes on which PAH have also direct effects The cytokines and activation of antigen presenting cells (APC: dendritic cells or macrophages/monocytes) stimulate the differentiation of T lymphocytes into a Th2 phenotype that is able to induce, in combination with cytokines, the activation and isotype switching of B lymphocytes (B), resulting in IgE production IgE could induce the release of mediators by mast cells (MC) and basophils (BP) leading to symptoms of acute asthma Furthermore, the infiltration of eosinophils (Eo) and their prolonged survival and activation by cytokines and IgE conduces to the release of mediators involved in the development of chronic asthma Neutrophil (NP) degranulation may lead to both acute and chronic asthma

could lead to symptoms of asthma, which have been shown to be aggravated after an increase in

PM10 levels (von Klot et al 2002)

This cytokine and chemokine expression is associated with the activation of upstream signaling cascades among which mitogen activated protein kinases (MAPKs) pathways have been shown to

be activated by particles DEP and their organic extracts increase the ERK 1/2 phosphorylation correlated to the GM-CSF release by 16HBE cell line as well as that of p38 (Bonvallot et al 2001) p38 activation has also been implicated in the IL-8 mRNA expression induced by DEP and their benzene organic extracts in BEAS-2B cell line (Kawasaki et al 2001) and in the release of IL-8 and RANTES induced by DEP extracts in peripheral blood mononuclear cells from allergic patients (Fahy et al 2000) Finally, JNK phosphorylation was observed in both HBE cells and macrophages exposed to DEP extracts (Li et al 2002) A more global overview of signaling pathways activation was obtained combining proteomic and phosphoproteins detection in HBE cells and macrophages exposed to crude or fractionated DEP extracts (Wang et al 2005) The p38 MAPK, JNK, and ERK cascades are activated mainly by a quinone-containing polar fraction and to a lesser extend by PAH-containing aromatic fraction

The expression of many inflammatory mediators is regulated by transcription factors among which the redox sensitive transcription factors NF-kB and AP-1 Whereas many studies have shown the activation of these transcription factors in particles treated-cells, few of them address the role of organic compounds In HBE cells, DEP and their extracts induced NF-kB (Bonvallot et al 2001; Kawasaki et al 2001), but not AP-1 activation (Bonvallot et al 2000)

Quinones

Quinones

DEP/PM

Carbonaceous core

ROS

Receptor

Signaling pathways activation ROS

CYP

CYP

Phase I enzymes

(CYP 1A1) Phase II enzymes (GST, NQO-1)

antioxidant enzymes (HO-1)

Cytokines: GM-CSF, IL-6 chemokines, IL-8, RANTES adhesion molecules (ICAM)

PAH

AhR

PAH-0-quinones

PAH

FIGURE 11.5 Scheme of the metabolic pathways activated by particles and the involvement of their organic component DEP, diesel exhaust particles; PAH, polyaromatic hydrocarbons; ROS, reactive oxygen species; AhR, aryl hydrocarbon receptor; CYP, cytochrome P450; XRE, xenobiotic responsive element; ARE, anti-oxidant responsive element; NRE, NF-kB responsive element; TRE, TPA responsive element