Particle Toxicology - Chapter 11 pot

11
Particle-Associated Organics
and ProinflammatorySignaling
Francelyne Marano, Sonja Boland, and Armelle
Baeza-Squiban
Laboratoire de CytophysiologieetToxicologie Cellulaire,
Universite
´
Paris 7–Denis Dide
`
rot
CONTENTS
11.1Introduction 211
11.2What Is the Role of These Organic Compounds in the Effects of PM? 211
11.2.1 The Particles OrganicFraction 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
11.3Conclusion 221
Abbreviations 221
References 221
11.1 INTRODUCTION
The burning of fossil fuels generates fine and ultrafineairborneparticles, which contain alarge
amount of organic compounds including polyaromatic hydrocarbons(PAH). These particlesmust
be takenintoaccount as they are considered to be among themostabundant componentsof
particulate matter 2.5 m m(PM
2.5
)inurban areas. Most of them are produced by diesel engine-
powered cars and diesel exhaustparticles (DEP) constitute, on average, 40% of the PM
10
in acity

such as Los Angeles (Diaz-Sanchez1997), and in akerbside station in Paris more than 50% of
particleswereclose to the ultrafine range ( % 0.26 m m), likely due to the influence of the traffic
(Baulig et al. 2004). Chemicalanalysis betweenPM
2.5
collectedinakerbside and abackground
station in Paris revealed that PAH are twice as important in the kerbside station. The resultsare
more relevantwith heavy PAH than light PAH,due to their higherstability. We have also observed
variationsofPAH according to the seasons, probably due to chemical reactions with atmospheric
oxidants.However, PAH are only apart of the organic component and they do notgreatly influence
the soluble organic fraction (SOF) measured after dichloromethane extraction that appeartobe
between10and 12% of the massofthe particles whatever the station and approximately 45% lower
than the SOF of DEP (20%)(Baulig et al. 2004).
11.2 WHATISTHE ROLE OF THESE ORGANIC COMPOUNDS
IN THE EFFECTS OF PM?
In this chapter, we do notconsider thegenotoxicand carcinogenic effects of theseorganic
compounds. Thedramatic increaseofhumanallergic airway diseasesinthe lastcentury has followed
211
© 2007 by Taylor & Francis Group, LLC
the increaseinthe use of fossil fuelsand manyepidemiological studies have provided indirect
evidence for acorrelation between particulate pollution andincreasedincidence of asthma and
allergicrhinitis.Numerous experimental studies in animals, in human volunteers, and in vitro
were performed to provide acausal explanationfor these observations. Diaz-Sanchez et al. have
publishednumerous studies on the roleofDEP and their associated PAH in the induction of allergic
airwaydiseases(Riedl and Diaz-Sanchez 2005). In vivo nasal provocation studies, usingamounts of
DEP equivalent to the total exposure in 1–3 days in Los Angeles, showed enhanced immunoglobulin
E(IgE) production in thehuman upperairway.Thisresponseappearstobelinkedtoorganic
compounds of DEP sinceithas been established that PAH–DEP could significantly increase IgE
mRNA andprotein production in IgE-secretingEpstein–Barr-virus transformedhuman Bcells
in vitro (Tsien et al. 1997). The effects of DEP were specific since they do not increaseIgG,IgA,
or IgM (Diaz-Sanchez et al. 1994). The ability of DEP to act as an adjuvant was tested by performing

nasalprovocationchallengeswithDEP, ragweedantigen Amba1 ,orbothsimultaneously in
ragweed-sensitive subjects(Diaz-Sanchez 1997).Ragweed-specific IgEwas 16 timeshigher
following ragweed plus DEPchallenge compared with ragweedalone.However,IgG levels
remained constant afterchallenge with DEPplusantigen. Furtherexperimentshaveshown an
increaseincytokine mRNAlevels 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 thereisahigh intraindividual
reproducibility of the nasal allergic responses in human exposure studies (Bastain et al. 2003).
Several candidategenes couldbeinvolved 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 adelayed response and does not
explain acute PM effects on airwayhyperreactivity. It has been recentlydemonstrated by Hao (Hao
et al. 2003), usingBALB/c mouse model sensitized by ovalbumin, that aerosolized DEP could
induceincreased airway hyperreactivity even if DEP deliveryisdelayed after the peak inflammatory
response. It was concluded that DEP induced airway hyperreactivity independently of adjuvant
effects. Interestingly, DEP co-administration with aneoallergen such as keyholelimpethemocyanin
showsthat the particles couldsynergize with the neoallergen and drivethe de novo production of
antigen-specific IgE (Diaz-Sanchezetal. 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 andthe provocation phase of allergybyinducingoxidative 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. Besidetheir adjuvant effect, these particleshave also an immunosuppressive
effect in animals. The increased susceptibility of the lung to infection in rats exposedtoDEP 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 amarker of
macrophage function, it was shownthat crude DEP organic extractsinhibit both Lipopolysac-
charide and Bacillus Calmette–Gue
´
rin (BCG) induced NO production by amurine macrophage
cell line explaining the impaired bacterial clearance noticed in aBCG mouselung infection model

(Saxena et al. 2003b). By fractionation of the organic extract,itappears that this inhibitoryeffect
was mainlydue to PAH and resin fractions (Saxena et al. 2003a).
Theidentification of the chemical componentsinvolved in thesebiological effects and the
understanding of theunderlyingmechanisms are still imperfect. Such studies are difficult,as
there exists agreatvariability in the chemical composition of PM according to their emission
sources, age, and site of sampling.
11.2.1 T HE P ARTICLES O RGANIC F RACTION
The organic fraction of particles comprises acountless quantityofcompounds (such as aliphatic
hydrocarbons, PAH, nitroaromatics hydrocarbons, quinones, aldehydes, and heterocyclics), some
Particle Toxicology212
© 2007 by Taylor & Francis Group, LLC
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 knownfamily of organic compounds adsorbed on particles, although they only represent afew
percent of the organic fraction. The interest in these compounds lies in their known genotoxic and
inflammatory properties and their use as atracer of source. They have been showntobeinhigher
concentrations in submicron particles (DeKok et al. 2005; Rehwagen et al. 2005), which can be
explained by the fact that soot from combustion sources consist primarily of fineparticles with high
PAH content and that the smaller particleshave arelatively 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, andcarcinogenesis(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 m g/g) and in Los Angeles PM
2.5
(5–730 pg/m
3
)(Cho et al. 2004).
11.2.2 B IOAVAILABILITY OF O RGANIC C OMPOUNDS

The presence on particles of organic compounds exhibiting apotential biological effect raises
the question of their bioavailablity. To understand the processes whereby particles deliver and
transfer toxic components to targetcells,experiments have been doneusing radio-labeled
benzo(a)pyrene(B(a)P)-bound denudedparticles.After their administration to dogs,the
extentand rate of release as wellastheir metabolic fate wereinvestigated(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,
asmaller fraction of B(a)P was slowly deposited but metabolized in the airway epithelium
(Gerde et al. 2001b). Nevertheless,alarge fraction of B(a)P remained bound to particleseven
6months after the exposure (Gerde et al. 2001b).
In cells, foreign substances are detoxifiedbytwo sequential reaction processes, namely, Phase I
and Phase II. In Phase Ireactions, 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 Imetabolites to more hydrophilic substances, allowing their elimination.
Among the members of the CYP gene family, CYP1 is knowntobeinduced by PAH through a
receptor-dependent mechanism. The cytosolic aryl hydrocarbon receptor (AhR), whenbound 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 CYP1A1expressionand activity
(Meek 1998; Bonvallot et al. 2001; Baulig et al. 2003a). As showninFigure11.1a, DEP and their
organic extract induceatransient CYP1A1mRNA expression in humanbronchial 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 aconcerted manner at the
transcriptional level through theantioxidant-responsiveelement(ARE)/electrophile-responsive
element. The transcription factor NF-E2-related factor-2 (Nrf2) is central to ARE-mediated gene
expression (Itohetal. 1997) andNrf2 ( K / K )miceexhibit significantreductionofphase II
enzymes (Cho et al. 2002). DEP inducethe translocation of Nrf2 to the nucleus of HBE cells,
increasenuclear proteinbinding to the ARE (Baulig et al. 2003a), as well as NQO1expression as
shown in Figure11.1b.

Whereas this biotransformation process aimed to detoxify xenobiotics, abioactivation may
occur and reactive metabolites are produced especially during the Phase I. By this way, PAH give
rise to electrophilic metabolitesresponsible for their genotoxicity.
Particle-Associated Organics and Proinflammatory Signaling 213
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Theobservation of the CYP1A1 gene induction in lung homogenates of Big Blue rats
exposed,byinhalation,towhole DEPfumes supports theargumentthatthe leachingof
organic compounds from particlescan occur (Sato et al. 2000). Moreover,inanother study,
it was shown that this transient CYP1A1induction in lungs of rats only occurswith DEP and
not with carbon black (Maand Ma 2002). Themechanismsoftransfer of organic compounds
from particlestothe target cells can involve the uptake of particles (Bonvallot et al. 2001).
However, arecent studyusing fresh butadiene soots suggests that the transfer to cells occurs
by the direct contact betweensoots and the plasma membrane, likelyinvolving apartitioning
mechanism (Penn et al. 2005).
Moreover, it is not known how effectively biological media (e.g., serumorinterstitial fluids)
can solubilize the organic compounds (Keane et al. 1991). It has been shown that the addition of
surfactant in an aqueoussuspension of DEP or carbon black particles on which aPAH mixture has
been previously adsorbed doesn’tfavor the leaching of PAH (Borm et al. 2005). In addition, the
PAH bioavailability is negligiblewhenthe PAH content is low relative to the particle monolayer
surface (Borm et al. 2005).
11.2.3 O RGANIC C OMPOUNDS AND O XIDATIVE S TRESS
Evidence for the involvementofoxidative stress in the effects of organic compounds camefrom
theinitial observationthatthe mortalityresultingfromlung edemaafter intratracheal
2h 6h 24 h48h
2.7
1.7
NQO1
DPL
DMSO
DEP

OE-DEP
CB
BaP
DPL
DMSO
DEP
OE-DEP
CB
BaP
DPL
DMSO
DEP
OE-DEP
CB
BaP
DPL
DMSO
DEP
OE-DEP
CB
BaP
18S
18S
6h 24 h48h
DPL
DMSO
DEP
OE-DEP
CB
BaP

DPL
DMSO
DEP
OE-DEP
CB
BaP
DPL
DMSO
DEP
OE-DEP
CB
BaP
DPL
DMSO
DEP
OE-DEP
CB
BaP
CYP1A1
18S
2h
(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 brespectively). Cells were treated or not with DEP (10 m g/cm
2
), carbon
black (10 m g/cm
2
)organic extracts of DEP (OE-DEP, 10 m g/mL) or benzo(a)pyrene (B(a)P ,3m M). RNA

(30 m g) were extracted from cells after 2, 6, 24, or 48 hoftreatment, electrophoresed, Northern-blotted, and
then incubated with a
32
P-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).
Particle Toxicology214
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administrationofwhole DEPintomice wassuppressed by pretreatmentwithpolyethylene
glycol-modified superoxide dismutase (Sagai et al. 1993) andthatitwas limited with
methanol-washed DEP. In this same study, it was shown that in acellularconditions, 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). Quinoneshave been reportedtoberesponsible for this radical production
due to their abilitytoundergoenzymatic(P450/P450 reductase) andnon enzymaticredox
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 irongives
%

OH by the Fentonreaction. A
methanol extract of DEP has been showntocause asignificant formation of O
$K
2
in the presence
of Cyt P450 reductase (Kumagai et al. 1997), an enzymewhich activity is increased in DEP-
treated mice (Lim et al. 1998). More recently, PM
2.5
have 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 thesesemiquinone radicals directly present on particles, others can be
produced during an alternative PAH-metabolizationinvolvingdihydrodiol dehydrogenase
leading to thegeneration of PAH o -quinones(Penning et al. 1999). Furthermore, the
CYP1A1 catalytic activity generatesreactive oxygen species(ROS) (Perretand Pompon
1998). Redox-active transition metals, redox cycling quinones, and PAH present on PM can
act synergistically to produceROS.
Taken altogether, thesedata reveal that organic compounds are asourceofROS. It explains
the pro-oxidant status measured using various specificfluorescent probesinairwayepithelial
cells and macrophages treatedeither with DEP, PM,ortheircorrespondingorganic extract,
whereascarbon black particles or solvent-extractedparticlesdonot have such an effect (Hiura
et al. 1999; Li et al. 2002; Baulig et al. 2003a; Baulig et al. 2004). Forexample, increased ROS
productiondetermined by thedichlorofluorescein fluorescence wasobserved in HBE cells
exposedfor 4hto DEP, urban PM
2.5
sampledinParis, and their respective extracts. Theextracts
gave afluorescence signal similar to native particles (Figure 11.2). Apro-oxidant status is known
to induce cellular specificresponses in the order that cells face oxidant insult. Various studies
have shownthatsuchresponses occurinDEP-treated cells. By agenomic approach,the
expression profiles of genes induced by organic extractsofDEP 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 amurine macrophages cell line (RAW264.7) exposed
to organic extracts of DEPaswellasinepithelialcells (Lietal. 2002).These data were
completed by observation of achange in the proteomeofRAW264.7 exposedtoDEP organic
extracts 51 proteins werenewly expressed butweresuppressedby N -acetylcysteine, athiol
antioxidant (Xiao et al. 2003).
Furthermore, from crude DEP extracts, Li and collaborators (Lietal. 2000)have shown
that only the polar fraction that is enrichedinquinones and the aromatic fraction enriched in
PAH were able to decrease the cellular GSH/GSSGratio in macrophages as well as to induce
HO-1 expression, bothindicative of asituationofoxidative stress. In otherrespects, the
comparison of coarse, fine, andultrafinePMrevealed that ultrafine PM have thehighest
redox activity(Cho et al. 2005), in agreement with the observation that ultrafines were the
most potent towardsinducingHO-1expressionand depleting intracellularGSH (Lietal.
2003). However, thesusceptibility to an adverse health effect of DEPislinkedtothe
functionalvariation in naturalantioxidant defenses.The polymorphism of GSTgenes has
been associated with atopyand experimental studies provide evidence that the GSTM1and
GSTP1 genotypes can play aroleinthe susceptibility to the adjuvant effect of DEP (Gilliland
et al. 2004).
Particle-Associated Organics and Proinflammatory Signaling 215
© 2007 by Taylor & Francis Group, LLC
11.2.4 O RGANIC C OMPOUNDS AND I NFLAMMATION
In vivo human exposure studies show that phenantrene, in contrasttocarbon black, increaseIgE
production, butdid notcause inflammatory cell infiltration(Saxonand Diaz-Sanchez2000).
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)ordichloromethane extraction with extracted DEP and carbon blackparticles
(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 exhibitingaSOF of 8.3% induce alower GM-CSF releasebyHBE cells than
DEPfromnon-equipped vehicles having a35% SOF(Figure 11.3b) (Bolandetal. 2000).
Concerning PM, until now, few studies have addressed the involvement of organic compounds.
By chemical fractionation (organic vs. aqueous fraction) of Paris urban PM
2.5
,itwas shown that the
GM-CSF secretion induced by native PM
2.5
in HBE cells was mimickedbytheir organic extracts
0
50
DCF fluorescence (% increase relative to control)
100
150
200
250
300
350
400
450
DEP OE-DEP PM OE-PM CB
FIGURE 11.2 Dichlorofluorescein (DCF) fluorescence intensity in human bronchial epithelial cells treated
with diesel exhaust particles (DEP, 10 m g/cm
2
)ortheir corresponding organic extract (OE-DEP), Paris urban

PM
2.5
(PM, 10 m g/cm
2
)ortheir correspondingorganic extract(OE-PM),orcarbonblackparticles (CB,
10 m g/cm
2
). Thecells were loaded with 2
0
,7
0
-dichlorofluorescein-diacetate (H2DCF-DA)at20 m Mfor
20 min and then treated or not with the toxics for 4h.The DCF fluorescence was measured by cytometry.
Results are expressed in %ofincrease of DCF fluorescence relative to control.
Particle Toxicology216
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whereasthe aqueousextract had aslight effect (Baeza-Squiban et al. 2005). The absence of effect of
the soluble fraction was alsoobserved with PM
10
(EHC-93) in normal HBE cells (Fujii et al. 2001).
The effect of urban particlesonthe cytokine production of macrophages was also due to the organic
fraction (Vogeletal. 2005) (Table 11.1).
The induction of chemokine release may be responsible for the inflammatory cell infiltration
observedinthe in vivo studies(Hiyoshietal. 2005).Figure11.4 showsthe inflammatory
response, which may result from leukocyterecruitment 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 Tlymphocytes (Bommel
et al. 2000)and organic extracts of DEP increase CD1a and costimulatory molecule expression on
monocyte derived dendritic cells (Koike and Kobayashi2005), IgE production of Blymphocytes
(Takenakaetal. 1995; Tsien et al. 1997),IL-4and histaminerelease frombasophils

(Devouassoux et al.2002) as well as mast cell (Diaz-Sanchez, Penichet-Garcia, andSaxon
2000)and eosinophil degranulation (Teradaetal. 1997). This releaseofgranulocyte mediators
100
80
60
40
20
pg of GM-CSF /mL
pg of GM-CSF /mL
0
C
(a)
(b)
DEP OE-DEP Extracted
DEP
Carbon
black
CDEP
-cata
OE-DEP
-cata
DEP
+cata
OE-DEP
+cata
100
120
140
80
60

40
20
0
FIGURE 11.3 GM-CSF release by HBE cells treated for 24 hwith (a) DEP, extracted DEP, or carbon black
particles at 10 m g/cm
2
and dichloromethane extracts of DEP (OE-DEP) at 20 m g/mL, (b) DEP collected from
adiesel engine with and without an oxidation catalyst and their corresponding organic extracts (OE-DEP).
* P ! 0.05compared with control value,
B
P ! 0.05compared with DEP-treated culture. (From Boland, S.,
Bonvallot,V., Fournier, T.,Baeza-Squiban,A., Aubier,M., andMarano, F., Am.J.Physiol.LungCell.
Mol. Physiol. 278, L25–L32, 2000. With permission).
Particle-Associated Organics and Proinflammatory Signaling 217
© 2007 by Taylor & Francis Group, LLC
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
eotaxin
Hiyoshi et al. (2005)
CB and organic extracts of DEP enhance ovalbuminspecific IgE and
IgG1
Heo, Saxon, and

Hankinson (2001)
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
RANTES
Kawasaki et al. (2001)
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
induced by allergen
Fahy et al. (2002)
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 Fujimakietal. (1997)
Tlymphocyte Pyrene increases IL-4 production Bommel et al. (2000)
(continued)
Particle Toxicology218
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Table 11.1 (Continued)
BlymphocyteOrganic extracts of DEP increase IgE production Tsien et al. (1997)
Organic extracts of DEP increase IgE production only in the presence
of IL-4 and CD40Ab
Takenaka et al. (1995)
Basophils Organic extracts of DEP increase IL-4 and histamine release in cells
from allergic and non-allergicsubjects
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,
RANTES)
Eo

MC
Epithelium
NP
MBP, ECP, EPO
Lipid mediators
Cytokines
Chronicasthma
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
Th2
B
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 Tlymphocytes into aTh2
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 prolongedsurvival and activation by cytokines and IgE conducestothe releaseofmediators
involved in the development of chronic asthma.Neutrophil(NP) degranulation may lead to bothacute
and chronic asthma.
Particle-Associated Organics and Proinflammatory Signaling 219
© 2007 by Taylor & Francis Group, LLC
couldlead to symptoms of asthma,which have been showntobeaggravated after an increase in
PM
10
levels (von Klot et al. 2002).
This cytokine and chemokine expressionisassociated 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 extractsincrease the ERK 1/2 phosphorylation
correlated to the GM-CSF releaseby16HBE cellline as well as that of p38 (Bonvallot et al. 2001).
p38 activation has alsobeen implicatedinthe IL-8 mRNA expression induced by DEP and their
benzene organic extracts in BEAS-2Bcellline (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
exposedtoDEP extracts (Lietal. 2002). Amore global overview of signaling pathwaysactivation
was obtainedcombining proteomic and phosphoproteins detection in HBE cells and macrophages
exposedtocrude or fractionated DEP extracts (Wang et al. 2005). Thep38 MAPK, JNK, and ERK
cascades are activated mainlybyaquinone-containingpolar fraction and to alesser extend by
PAH-containingaromatic fraction.
Theexpression of manyinflammatory mediatorsisregulated by transcription factorsamong
which the redox sensitive transcription factorsNF-k Band AP-1. Whereas many studies have shown
the activation of thesetranscription factorsinparticlestreated-cells, few of them address the roleof
organic compounds. In HBE cells, DEP and their extracts induced NF-k B(Bonvallot et al. 2001;
Kawasaki et al. 2001), but not AP-1activation (Bonvallot et al. 2000).
Quinones
Quinones
DEP/PM
Carbonaceous
core
ROS
Receptor
Signaling pathways activation
ROS
CYP
CYP
Phase Ienzymes
(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
XRE ARE NRE TRE
PAH
PAH-0-
quinones
NF-κ B
AP-1
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.
Particle Toxicology220
© 2007 by Taylor & Francis Group, LLC
11.3 CONCLUSION
It is now clear that organic compounds play an essential roleinthe adverse effects of atmospheric
particles.Mostofthe studieswere performed on DEP, which were used as amodel forPM
containing xenobiotics such as PAHand quinones. Thecomparisonofthe effectsinduced by
DEP, organic extractsofDEP, and extracted DEP revealed that organic compounds are mainly
involved in the proinflammatoryresponse. Theextend of the response depends on the bioavail-
abilityofthese compounds that couldbeextracted by broncho-alveolar fluidorafterthe

internalization of particles by the airway epithelium andthe macrophages.The metabolism of
these molecules generates ROS that could be responsible for the activation of signaling pathways
and redox-sensitive transcription factorssuch as NF-k Band AP1. Thus, cytokine genes under the
control of thesefactors couldinitiate transcription. Moreover, recent data have shownthat DEP and
PM couldupregulate the expression of growth factorssuchasamphiregulin,aligandofthe
epidermal growth factorreceptor in humanHBE,and increase its secretion(Blanchet et al.
2004). Amphiregulin, which could have autocrine as well as paracrine effects, was demonstrated
to be involved in cytokine secretion and would participate to amore general cellular response for
sustaining the proinflammatoryeffects of particles. All of these results, which are summarized in
Figure 11.5,provide amechanistic explanation for the in vivo observations of animal or human
responses to DEP or PM exposures and to epidemiological studies.
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ABBREVIATIONS
AhR: Aryl hydrocarbon receptor
ARE: Antioxidant-responsive element
BCG: Bacillus Calmette–Gue
´
rin
B(a)P: Benzo(a)pyrene
CYP: Cytochrome P
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PM
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:Particulate matter
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SOF: Soluble organic fraction
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