# Chapter 5. Possible Pa thways/ Modes of Action

The mechanisms underlying pulmonary effects of inhaled PM have been well-studied in the laboratory and there is general agreement regardin g the key roles played by cellular injury and inflammation. These pathways are initiated fo llowing the deposition of inhaled particles on respiratory tract surfaces. Since most of these studies were conducted at concentrations of PM much higher than ambient levels, there is some question regarding the relevance of these responses and mechanisms to ambient exposures. Interestingly, inhaled PM may also affect the cardiovascular, hematopoietic and other systems. Mechanisms underlying these extra-pulmonary effects are incompletely understood. However, pulmonary inflammation can lead to systemic infl ammation and pulmonary reflexes can activate the autonomic nervous system (ANS). These latter re sponses may mediate cardiovascular and other systemic effects, as will be discussed below. In addition, it has been proposed that PM or soluble components of PM reach the circulation by tra nslocating across the epithelial and endothelial barriers of the respiratory tract. In this way, PM or its components may interact directly with cells in the vasculature and blood and be transported to the heart and other organs. At this time, evidence clearly supports the translocation of small solutes following inhalation exposures and the translocation of soluble components of PM following some high dose exposures involving intratracheal (IT) instillation. However, there is insufficient evidence to support translocation of appreciable amounts of intact particles followi ng inhalation exposures at lower concentrations (Section 4.3.3.1). Future studies will be required to resolve these issues. The following sections discuss biological pathwa ys which comprise proposed modes of action for the pulmonary and extra-pulmonary effects of inhaled PM. Overall themes are emphasized and supportive evidence from new in vitro and in vivo animal studies is cited. The characterization of evidence here is for PM in general, since most of the potential pathways or modes of action do not appear to be specific to a particular size class of PM. However, characteristics of ultrafine particles (UFPs) may allow for unique modes of action or ef fects disproportionate to their mass, as will be described below. Recent studies suggest an enhanc ed potential of this size class of PM to cause adverse effects; however evidence supporting this hypothesis is limited. Finally, a compilation of results from new inhalation studies which are releva nt to ambient PM exposures and which confirm and extend these proposed mechanisms is found at th e end of this chapter. Detailed descriptions of these key new studies are found in Chapters 6 and 7.



# 5.1. Pulmonary Effects

5.1.1. Reactive Oxygen Species

A great deal of research interest has focused on the role of reactive oxygen species (ROS) in the initiation of pulmonary injury and inflammation following exposure to PM. Numerous studies have demonstrated PM oxidative potential in in vitro and in vivo assay systems (Ayres et al., 2008, 155666 ; Cho et al., 2005, 087937 ; Shi et al., 2003, 088248 ; Tao et al., 2003, 156111 ). Both redox active surface components, such as metals and organic species, and the surface characteristics of crystal structures have been shown to contri bute to oxidative potential (Jiang et al., 2008, 156609 ; Tao et al., 2003, 156111 ; Warheit et al., 2007, 090482 ). In this way, PM may be a direct source of ROS in the respiratory tract ( Figure 5-1 ).

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and

Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific literature used by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA) and the Integrated Risk Information System (IRIS)

.

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### Figure 5-1. PM oxidative potential.

PM may also act as an indirect source of ROS in the respiratory tract by stimulating cells to produce ROS (Ayres et al., 2008, 155666 ; Tao et al., 2003, 156111 ) ( Figure 5-2 ). This may explain the observation that the oxidative potential of isolat ed PM does not always correlate with cellular or tissue oxidative stress induced by PM exposure. Ex posure to PM increases intracellular production of ROS by a variety of mechanisms. For example, PM interaction with cell surfaces results in stimulation of NADPH oxidase in macrophages (i.e., the respiratory burst) (Dostert et al., 2008, 155753 ) and in epithelial cells (Amara et al., 2007, 156212 ; Becher et al., 2007, 097125 ; Tamaoki et al., 2004, 157040 ). Absorption of PM soluble component s (e.g., PAH, transition metals) by respiratory tract cells can occur (Penn et al., 2005, 088257 ) and be followed by microsomal transformation of PAHs to quinones or by redox cycl ing of transition metals with production of intracellular ROS (Molinelli et al., 2002, 035347 ; Xia et al., 2004, 087486 ). Disruption of intracellular iron homeostasis with the subsequent generation of ROS has also been demonstrated following PM exposure (Ghio and Cohen, 2005, 088272 ). In some cases, mitochondria serve as the source of ROS in response to PM (Huang et al., 2003, 156573 ; Risom and Loft, 2005, 089070 ; Soberanes et al., 2006, 156991 ; Soberanes et al., 2009, 190483 ). Furthermore, PM interaction with cells can lead to the induction of nitric oxide synthase (Becher et al., 2007, 097125 ; Lindbom et al., 2007, 155934 ; Xiao et al., 2005, 156164 ; Zhao et al., 2006, 100996 ) and the production of nitric oxide and other reactive nitrogen species (RNS).

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### Figure 5-2 PM stimulates pulmonary cells to produce ROS/RNS.

Although all size fractions of PM may contribute to oxidative and nitrosative stress, UFPs may contribute disproportionately to their mass due to their large surface/volume ratio. The relative enrichment of redox active surface components, such as metals and organics, per unit mass may translate to a relatively greater oxidative potential of UFPs compared with larger particles with similar surface components. In addition, the greater surface per unit volume could potentially deliver relatively more adsorbed soluble components to cells. These components may undergo intracellular redox cycling following cellular uptake. Furtherm ore, per unit mass, UFPs may have more opportunity to interact with cell surfaces due to th eir greater surface area and their greater particle number compared with larger PM. These interacti ons with cell surfaces can lead to ROS generation, as described above. Recent studies have also demonstrated that UFPs have the capacity to cross cellular membranes by non-endocytotic mechanisms involving adhesive interactions and diffusion (Geiser et al., 2005, 087362 ), as described in Chapter 4. This may allow UFPs to interact with or penetrate intracellular organelles. In general, high levels of intracellular ROS/RNS can lead to irreversible protein modifications, loss of cellular membrane integrity, DNA damage and cellular toxicity. Lower levels of ROS/RNS may cause reversible protein modifications that trigger intracellular signaling pathways and/or adaptive responses. Thus PM-dependent generation of ROS may be responsible for a continuum of responses from cell signaling to cellular injury.



## 5.1.2. Activation of Cell Signaling Pathways

Activation of cell signaling pathways by RO S/RNS has received increasing attention by numerous investigators over the years. An early example was provided by Kaul and Forman (1996, 155892 ) who demonstrated that respiratory burst-derived H 2 O 2 activates the transcription factor

December 2009 5-3nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κ B). Numerous studies since then have demonstrated that PM, which serves as both a direct and indirect source of ROS/RNS, activates cell signaling pathways by this mechanism. PM also has the potential to activate cell signaling by mechanisms that are independent of ROS/RNS. For example, PM delivers water-soluble components, such as endotoxin and zinc (Zn), to cell surfaces. Endotoxin binds to toll-like receptors on alveolar macrophages and other cells, resulting in the upregulation of cytokines (Becker et al., 2002, 052419 ). Zn, a transition metal which does not redox cycle, inhibits protein tyrosine phophatases in airway epithelial cells resulting in a cascade of cell signaling events (Tal et al., 2006, 108588 ). Similarly, PM-mediated delivery of lipid soluble components such as PAH results in binding and activation of the arylhydrocarbon receptor (AhR). AhR is a transcription factor responsible for the upregulation of CYP1A1, a cytochrome oxidase involved in PAH biotransformation to metabolites capable of forming DNA adducts or eliciting oxidative stress responses (Rouse et al., 2008, 156930 ). In addition, interaction of PM with cell surfaces may activate cell signaling by pert urbation of the cytoskeleton, adherence, internalization, or rece ptor-mediated pathways. Recent studies involving PM exposures have focused on intracellular pathways involving protein kinases, such as mitogen-activated protein kinase (MAPK) (Bayram et al., 2006, 088439 ; Lee et al., 2005, 156682 ; Roberts et al., 2003, 156051 ; Soberanes et al., 2009, 190483 ); AKT (Ahsan et al., 2005, 156200 ); src (Cao et al., 2007, 156322 ) and epidermal growth factor receptor (Blanchet et al., 2004, 087982 ; Cao et al., 2007, 156322 ; Tamaoki et al., 2004, 157040 ), as well as ras (Tamaoki et al., 2004, 157040 ), toll-like receptors (Becker et al., 2002, 052419 ; Becker et al., 2005, 088590 ), protein tyrosine phosphatases (Tal et al., 2006, 108588 ), phospholipases A 2 (Lee et al., 2003, 156678 ), calcium (Agopyan et al., 2003, 155649 ; Brown et al., 2004, 155705 ; Brown et al., 2004, 088663 ; Geng et al., 2005, 096689 ; 2006, 097026 ; Sakamoto et al., 2007, 096282 ), caspases (Soberanes et al., 2006, 156991 ; Zhang et al., 2007, 156179 ), poly (ADP-ribose) polymerase family member 1 (PARP-1) (Zhang et al., 2007, 156179 ) and histone acetylation (Gilmour et al., 2003, 096959 ). The transcription factors regula ted by these pathways, including NF- κ B (Bayram et al., 2006, 088439 ; Lee et al., 2005, 156682 ; Takizawa et al., 2003, 157039 ), activator protein 1 (AP-1) (Donaldson et al., 2003, 156408 ), signal transducers and activators of transcription protein (STAT) (Cao et al., 2007, 156322 ), antioxidant response element (ARE) (Li and Nel, 2006, 156694 ), and AhR (Rouse et al., 2008, 156930 ) have also been studied following PM exposures. Activation of these intracellular pathways and transcription factors leads to the upregulation of genes responsible for inflammatory, immune and acute phase responses as well as genes responsible for antioxidant defense and xenobiotic metabolism.



## 5.1.3. Pulmonary Inflammation

Following PM exposure, transcription factor ac tivation in macrophages and epithelial cells stimulates the synthesis and release of soluble me diators involved in inflammatory and immune responses including cytokines, chemokines, proteases and eicosanoids ( Figure 5-3 ). These substances play a role in recruiting inflammatory cells such as neutrophils, monocytes, mast cells and eosinophils to the lung. Interactions between macrophages and epithelial cells enhance these responses (Tao and Kobzik, 2002, 157044 ).

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### Figure 5-3. PM activates cell signaling pathways leading to pulmonary inflammation.

Inflammatory cells can serve as a source of extracellular ROS which, along with soluble mediators derived from the inflammatory cells, amplify the inflammatory response. Unchecked inflammation may cause cellular and tissue injury through the generation of excess amounts of ROS and soluble mediators. In some cases the oxidative pot ential of PM is well-correlated with the degree of inflammation (Dick et al., 2003, 036605 ), suggesting that the inflammation is a direct consequence of PM-generated ROS. However, in other cases the oxidative potential of PM is not well-correlated with the degree of inflammation (Beck-Speier et al., 2005, 156262 ), suggesting that the inflammation is a consequence of the ROS-independent mechanisms by which PM activates intracellular signaling pathways. Par ticle surface area has been identified as a key determinant of the extent of inflammation in the case of low-toxici ty, low-solubility particles (Donaldson et al., 2008,

December 2009 5-5190217 ). Moreover, UFPs may cause inflammation di sproportionately to their mass compared with PM of larger sizes given their large surface/volume ratio compared with other PM fractions. PM exposure often results in neutrophilic infla mmation in laboratory studies (Tao et al., 2003, 156111 ). Neutrophilic inflammation is also associated with acute lung injury in humans as well as chronic lung diseases such as COPD and ce rtain forms of asthma (Barnes, 2007, 191139 ; Cowburn et al., 2008, 191142 ). Circulating neutrophils respond to chemotactic factors in the lung such as leukotriene B 4 and IL-8 (Barnes, 2007, 191139 ). They migrate into the lung parenchyma across the pulmonary capillary network and into the airways from the bronchial circulation (Cowburn et al., 2008, 191142 ). As a consequence of priming by infl ammatory mediators or contact with extracellular matrix components, neutrophils be come hyperresponsive to activating signals and insensitive to chemotactic si gnals (Cowburn et al., 2008, 191142 ). Activation results in neutrophil degranulation, respiratory burst responses and soluble mediator release (Cowburn et al., 2008, 191142 ). Neutrophils eventually undergo apoptosis and are phagocytized by inflammatory macrophages (Cowburn et al., 2008, 191142 ). This is accompanied by the release of antiinflammatory mediators such as IL-10 and transforming growth factor- β (TGF- β ) (Cowburn et al., 2008, 191142 ). These steps are key to the resolution of inflammation and prevent unregulated release of toxic neutrophil products such as neutrophil elastase (Cowburn et al., 2008, 191142 ). Thus, circumstances leading to the decreased ingestion of apoptotic neutrophils by macrophages may lead to tissue injury. Impairment of macrophage functi on may serve as an important mechanism by which PM contributes to disease.



## 5.1.4. Respiratory Tract Barrier Function

Epithelial injury can lead to an increase in permeability of the airway epithelial and alveolar-capillary barriers (Braude et al., 1986, 155701 ). Enhanced transport of soluble and possibly of insoluble PM components into the circulat ion may occur under these conditions. Increased epithelial permeability is also associated with enhanced immune responses to proteins, including allergens, on the epithelial surface, presumably due to the greater availability of antigens to underlying immune cells (Wan et al., 1999, 191903 ). Furthermore, endothelial injury can compromise the integrity of the al veolar-capillary barrier resulting in transvascular fluid and solute flux (Braude et al., 1986, 155701 ). Soluble mediators derived from inflammatory and lung cells (Chang et al., 2005, 097776 ) and peptides released by some nerve cells (Widdicombe and Lee, 2001, 019049 ) can increase the permeability of the alveolar-capillary barrier and result in alveolar edema. Compromised barrier function in the airways ma y lead to airway edema. Edema occurring secondarily to nerve cell stimulation is one component of the process termed neurogenic inflammation. Given the small size of UFPs, modest changes in epithelial permeability may particularly affect the disposition of this fraction. Enhanced tra nslocation to interstitial compartments or to the circulation may be important sequelae. A recent study described in Section 4.3.4.3 demonstrated greater translocation of UFPs compared with PM 2.5 into the circulation of rodents treated with endotoxin to induce acute lung injury prior to IT instillation of PM (Chen et al., 2006, 147267 ). Furthermore, epithelial injury in another model re sulted in greater translocation of UFPs into the interstitial compartment (Adamson and Prieditis, 1995, 189982 ).



## 5.1.5. Antioxidant Defenses an d Adaptive Responses

Antioxidant defenses and adaptive responses are important modulators of oxidative stress and other cellular stresses resulting from PM exposure. Antioxidants are present in the epithelial lining fluid in all regions of the respiratory tract. In addition, they are present in cells of the lung parenchyma and inflammatory cells found in airwa ys and alveoli. Some antioxidants act directly against oxidant species (e.g., glutathione, ascorb ate, superoxide dismutase) while others act indirectly (e.g., gamma-glutamylcysteine synthetase [ γ GCS], glutathione reductase). Furthermore, some antioxidants (e.g., Phase 2 enzyme s heme oxygenase-1[H O-1], NADPH quinone oxidoreductase 1 [NQO1], glutathi one-S-transferase [GST]) are i nducible via activation of the nuclear factor (erythroid-derived 2)- related factor 2 (Nrf2)-ARE pathway, which occurs as an adaptive response to stress (Cho et al., 2006, 156345 ; Li and Nel, 2006, 156694 ). Antioxidants play

December 2009 5-6an important role in reducing the oxidative potential of those PM species that directly generate ROS. They also inhibit responses due to generation of intracellular ROS. Recently a three-tier response to oxidativ e stress was proposed (Li and Nel, 2006, 156694 ). In this scheme, mild oxidative stress enhances anti oxidant defenses by upregulating Phase 2 and other antioxidant enzymes (Tier 1). Further increase in oxidative stress induces inflammation (Tier 2) and cell death (Tier 3). Experimental evidence is supp ortive of this scheme. Numerous studies have demonstrated that enhancement of lung and cellula r antioxidant defenses inhibits inflammation, cytotoxicity and other responses following exposure to PM (Ahsan et al., 2005, 156200 ; Bachoual et al., 2007, 155667 ; Bayram et al., 2006, 088439 ; Chang et al., 2005, 097776 ; Imrich et al., 2007, 155859 ; Koike and Kobayashi, 2005, 088303 ; Koike et al., 2004, 058555 ; Li et al., 2007, 155929 ; Ramage and Guy, 2004, 055640 ; Rhoden et al., 2004, 087969 ; Steerenberg et al., 2004, 087981 ; Takizawa et al., 2003, 157039 ; Tao et al., 2003, 156111 ; Upadhyay et al., 2003, 097370 ; Wan and Diaz-Sanchez, 2006, 097399 ; Wan and Diaz-Sanchez, 2007, 156145 ; Yin et al., 2004, 087983 ). Cellular and tissue exposure to xenobiotics carried by PM can lead to induction of Phase 1 and Phase 2 detoxifying enzymes following the activation of cell signaling pathways and transcription factors AhR and ARE, respectively (Rengasamy et al., 2003, 156907 ; Rouse et al., 2008, 156930 ; Zhao et al., 2006, 100996 ).



### Figure 5-4. Potential pathways for the effects of PM on the respiratory system.

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## 5.1.6. Pulmonary Function

PM exposure may alter pulmonary function by a variety of different mechanisms ( Figure 5-4 ). In the short-term, airway hyperresponsiveness (AHR) may ensue due to the influence of inflammatory mediators. In the long-term, morpholo gical changes may occur, in some cases leading to mucus hypersecretion and airway remodeling. Activ ation of irritant receptors and stimulation of the ANS in the respiratory tract is another mechanism by which PM exposure may alter pulmonary function (Section 5.4 ).



## 5.1.7. Allergic Disorders

PM exposure sometimes leads to the development of allergic immune responses ( Figure 5-4 ). These responses are predominately mediated by T helper 2 cells (Th2). Th1 responses, characterized by IFN- γ and classical macrophage activation, are inflammatory; in excess they can lead to tissue damage. Alternatively, Th2 responses are associated with allergy and asthma and are characterized by IL-4, IL-5, IL-13, influx of eosinophils, B–lymphocyte production of IgE, and alternative macrophage activation. PM exposure can also lead to the exacerbation of airway allergic responses, such as antigen-specific IgE production and AHR. Due to soluble mediators and immune cell trafficking, pulmonary exposure may result in systemic immune alterations. Not only do macrophages ingest PM, but they are also antigen presenting cells whose level of activation dict ates costimulation and thus subsequent T cell responses. These cells are highly mobile and can transport PM to other sites such as lymph nodes. Dendritic cells (DC) also play a key role as antig en presenting cells and in modulating T and B cell activity. A cell culture model of the human epithe lial airway wall was used to demonstrate that DC extended processes between epithelial cells through the tight junctions, collected particles in the lumenal space and transported them across the epithelium (Blank et al., 2007, 096521 ). DC also transmigrated through the epithelium to take up particles on the epithelial surface (Blank et al., 2007, 096521 ). Furthermore, DC interacted with particle -loaded macrophages on top of the epithelium and with other DC within or beneath the epithelium to transfer particles (Blank et al., 2007, 096521 ). In vitro studies also demonstrated that the adjuvant activity of diesel exhaust particles (DEPs) involved stimulation of immature monocyte-derived dendritic cells (iMDDC) to undergo maturation in response to an altered airway epithelial cell-de rived microenvironment (Bleck et al., 2006, 096560 ). Additionally, DEP directly influenced the prof ile of cytokines secreted by DC and caused a predisposition toward Th2-mediated or allergic responses (Chan et al., 2006, 097468 ). Thus PM can negatively affect both innate immunity through effects on macrophage pathogen handling (Section 5.1.8 ) as well as adaptive immunity by altering macrophage or DC antigen presenting activity and subsequent T cell responses. Moreover, recent studies have demonstrated th at ambient PM can act as an adjuvant for allergic sensitization with UFPs having a greater ef fect than fine particles (de Haar et al., 2006, 144746 ; Li et al., 2009, 190457 ). This has been attributed to higher oxidative potential of the UFPs compared with the same mass of par ticles of larger size (Li et al., 2009, 190457 ), although the larger surface area and particle number per unit mass as we ll as the propensity of UFPs for trans-epithelial movement may also contribute to this effect.



## 5.1.8. Impaired Lung Defense Mechanisms

PM exposure may impair lung defense mechanisms and result in frequent or persistent infections ( Figure 5-4 ). Potential targets include mucocili ary transport, surfactant function and pathogen clearance. Pathogen clearance is dependent on the integrity of macrophages and their migration, phagocytosis and respiratory burst functions. PM-mediated cytotoxicity of macrophages with the concomitant release of lysosomal contents may affect pathogen clea rance and cause damage to nearby cells and tissues. IT instillation and cell culture experiments have demonstrated PM-dependent impairment of lung defense mechanisms (Jaspers et al., 2005, 088115 ; Kaan and Hegele, 2003, 095753 ; Long et al., 2005, 087454 ; Moller et al., 2005, 156770 ; Monn et al., 2003, 052418 ; Roberts et al., 2007, 097623 ; Yin et al., 2004, 087983 ).

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## 5.1.9. Resolution of Inflammation/P rogression or Exacerbation of Disease

Resolution of pulmonary inflammation and injury has been demonstrated in many experimental models using higher than ambient concentrations of PM. Factors contributing to this complex process are likely to include the uptake an d clearance of PM by macrophages; the retention of PM in parenchymal cells and tissues; the bala nce of pro/anti-inflammatory soluble mediators, oxidants/antioxidants and proteases/anti-proteases; and the presence of pre-existing disease. These factors may also influence the resolution of pulmonary responses to ambient PM exposures (Figure 5-4). The long-term consequences of prolonged inflammation are not likely to be beneficial and may lead to remodeling of the respiratory tract and to the progression or exacerbation of disease.



## 5.1. 9.1. Fact ors Affecting the Retention of PM

Clearance of poorly soluble particles from ET, TB and alveolar regions is extensively discussed in Section 4.3. While clearance from ET and TB regions generally occurs over hours to days, clearance from the alveolar compartment is much slower, occurring over months to years depending on the species. Phagocytosis by alveol ar macrophages and transport by the mucociliary escalator is the primary mechanism of clearance from the alveolar compartment although neutrophil phagocytosis also plays a role (Snipes et al., 1997, 156092 ). Pre-existing disease can alter the extent and localization of PM deposition as discussed in Section 4.2.4.5. In addition, mechanisms of clearance may be altered in cases of pre-existing di sease as discussed in Section 4.3.4.3. While mild asthma was associated with enhanced mucociliary clearance, acute lung injury was associated with enhanced particle translocation to the circulation and the interstitial compartment. Whether retained particles are localized in alveolar macrophages or parenchymal tissue also differs according to species (Snipes, 1996, 076041 ). UFPs may have a special propensity for retenti on given the decreased efficiency of alveolar macrophages for phagocytosis of this size particle (Oberdörster, 1988, 006857 ) and the demonstration that UFPs can readily cross cellular membranes (Geiser et al., 2005, 087362 ). Some studies suggest that UFPs are taken up by epithelial cells and move to the interstitum where they are cleared by other pathways (Semmler-Behnke et al., 2007, 156080 ); however clearance mechanisms are not entirely understood. Enhanced deposition of particles in “hot spot s” may influence retention. For example, deposition in the centriacinar or proximal alveolar region, where clearance is slow, may result in accumulated particle dose in this region and the potential for prolonged inflammation at the site leading to the development of pulmonary fibrosis or emphysema (Donaldson et al., 2008, 190217 ). A recent study suggests an important role for retained particles in the progression of disease. Complexation of endogenous iron by retained particles resulted in retained particles growing larger over time. The authors suggested that redox cycling of complexed iron may be responsible for disease progression (Ghio and Cohen, 2005, 088272 ; Ghio et al., 2004, 155790 ).



## 5.1. 9.2. Fact ors Affecting the Balance of Pro/Anti-Inflammatory Mediators, Oxidants/Anti-Oxidants and Proteases/Anti-Proteases

Inflammation can be enhanced by pro-inflammatory mediators or dampened by antiinflammatory mediators. Production of anti-inflamma tory mediators normally occurs at several steps of the inflammation pathway, such as the release of IL-10 and TGF- β during phagocytosis of apoptotic neutrophils by macrophages (Cowburn et al., 2008, 191142 ). Dsyregulation of the inflammatory process may prevent the resolution of inflammation. PM exposure may result in the production of pro-inflammatory mediators as well as decrease the production of anti-inflammatory mediators by impairing macrophage function. An unfavorable balance of oxidants to antioxidant s in the lung is associated with inflammatory lung diseases including asthma a nd COPD (Rahman et al., 2006, 191165 ). PM is likely to contribute to an unfavorable balance through its oxidative potential and capacity to promote cellular production of ROS. Exacerbations of asthma and COPD resulting from bacteria l and viral infections are also associated with increased oxi dative stress (Barnes, 2007, 191139 ). Conversely, antioxidants may reduce neutrophilic inflammation associated with oxidative stress (Barnes, 2007, 191139 ).

December 2009 5-9Protease/anti-protease balance has long been tie d to the pathogenesis of emphysema and other forms of COPD (Owen, 2008, 191162 ). Key steps include the release of proteinases by inflammatory cells which degrade the extracellular matrix components of alveolar walls. Destruction of alveolar walls and airspace enlargement ensues. Endogenous anti-protease defenses in the lung modulate this response but may be insufficient to prevent it dur ing prolonged inflammation. Proteases also play a role in pathologies which lead to small airway fibrosis (Owen, 2008, 191162 ). Oxidative stress has been linked to both activation of proteases and inactivation of anti-proteases (Owen, 2008, 191162 ). PM may contribute to an unfavorable protease/anti- protease balance through the generation of ROS. Although there are numerous inflammatory cell-de rived proteases and lung anti-proteases, many recent studies have focused on matrix meta lloproteinases (MMPs). MMPs are a family of Zncontaining enzymes normally found in an inactive pro-enzyme form. Activation involves proteolytic cleavage or oxidation of the “cysteine switch” (Pardo and Selman, 2005, 191163 ). Inhibitors include tissue inhibitors of metalloproteinases (TIMPs) (Pardo and Selman, 2005, 191163 ). In particular, MMP-1 is well-studied and found to play an im portant role in physiological processes such as development and wound repair as well as in diseases such as pulmonary emphysema, fibrosis, asthma and bronchial carcinoma (Li et al., 2009, 190424 ; Pardo and Selman, 2005, 191163 ). In addition to its activity in degrading collagenase, MMP-1 also acts on non-matrix substrates and cell surface molecules suggesting that it may influence cell signaling (Pardo and Selman, 2005, 191163 ). MMP-2 and MMP-9 are thought to be involved in the pathogenesis of disease through their gelatinase activity. Interestingly, recent in vitr o studies have demonstrated upregulation of MMP-1 by hydrogen peroxide, cigarette smoke and DEPs (Amara et al., 2007, 156212 ; Li et al., 2009, 190424 ; Mercer et al., 2004, 191180 ), and up-regulation of MMP-12 following instillation of PM collected from the Paris subway (Bachoual et al., 2007, 155667 ). These considerations suggest that particulate air pollution may act via MMP to mediate progression or exacerbation of lung disease.



## 5.1. 9.3. Pre- Existing Disease

In addition to its effects on deposition, reten tion and clearance of PM described above, preexisting disease may also alter the balance of th e aforementioned factors. For example, acute exacerbations of COPD are characterized by a rapid influx of neutrophils into the airways (Owen, 2008, 191162 ). However, clearance of apoptotic neutrophi ls by macrophages is impaired in COPD leading to greater release of neutrophil-derived inflammatory mediators, oxidants and proteases (Owen, 2008, 191162 ). Thus, exacerbation of disease may occur as a result of unchecked inflammation.



## 5.1.10. Pulmonary DNA Damage

Pulmonary DNA damage can occur primarily or secondarily to PM exposure. Primary effects include oxidative DNA injury or DNA adduct formation due directly to PM while secondary effects occur due to PM-mediated inflammation (De Kok et al., 2005, 088656 ; Gabelová et al., 2007, 156457 ; Gallagher et al., 2003, 140171 ; Schins and Knaapen, 2007, 156074 ). These responses may lead to chromosomal aberrations or DNA strand breaks. PM effects on cell cycle arrest, proliferation, apoptosis, and DNA repair mechanisms may also in fluence the genotoxic, mutagenic or carcinogenic potential of DNA damage as reviewed by Schins et al. (2007, 156074 ).



## 5.1.11. Epigenetic Changes

Epigenetic mechanisms regulate the transcript ion of genes without altering the nucleotide sequence of DNA. These mechanisms generally involve DNA methylation and histone modifications, leading to alterations which may have long-term consequences or are heritable (Jones and Baylin, 2007, 191153 ; Keverne and Curley, 2008, 191154 ). DNA methylation and histone modifications, which include methylation, acet ylation, phosphorylation, ubiquitylation and sumoylation, are known to be linked (Hitchler and Domann, 2007, 191151 ; Jones and Baylin, 2007, 191153 ). Numerous studies have identified epigenetic pr ocesses in the control of cancer (Foley et al., 2009, 191144 ; Gopalakrishnan et al., 2008, 191147 ; Jones and Baylin, 2007, 191153 ; Valinluck et al., 2004, 191170 ), embryonic development (Foley et al., 2009, 191144 ; Gopalakrishnan et al., 2008,

December 2009 5-10191147 ; Keverne and Curley, 2008, 191154 ) and inflammation and other immune system functions (Adcock et al., 2007, 191178 ). Epigenetic modifications resulting in decreased expression of tumor suppressor genes and increased expression of transforming genes have been observed in human tumors (Valinluck et al., 2004, 191170 ). In general, transcription repression is associated with DNA methylation in promoter regions of genes. Cytosine methylation in CpG dinu cleotides has emerged as an important, heritable epigenetic modification which can result in chromatin remodeling and decreased gene expression (Valinluck et al., 2004, 191170 ). Global changes in DNA methylation are also seen in cancer and hypomethylation is associated with genomic instability (Gopalakrishnan et al., 2008, 191147 ). Embryonic development is characterized by several phases of epigenetic modifications. DNA methylation is very dynamic following fertilizatio n, with demethylation and re-methylation of egg and sperm genomes occurring immediately (Foley et al., 2009, 191144 ). Imprinted genes, however, retain the methylation profile of the pa rent of origin (Foley et al., 2009, 191144 ). Epigenetic changes accumulated through a life course may be passed from parent to offspring in the germline (i.e., germline transmission of epimutation) if they surv ive the epigenetic remodeling that occurs during gametogenesis and early embryogenesis (Foley et al., 2009, 191144 ). Early development is characterized by the process of cell differentiation, which produces different cell types and involves the selective activation of some sets of genes and th e silencing of others in a temporal pattern (Foley et al., 2009, 191144 ; Gopalakrishnan et al., 2008, 191147 ). DNA methylation is postulated to provide a basis for cell differentiati on (Gopalakrishnan et al., 2008, 191147 ). In the lung, histone acetylation and methylat ion have been linked to inflammatory gene expression, T cell differentiati on, and the regulation of macrophage function following pathogen challenge (Adcock et al., 2007, 191178 ). Furthermore, altered patterns of methylation and acetylation have been reported in inflammatory diseases (Adcock et al., 2007, 191178 ). Reduced expression and activity of histone deacetylase have been demonstrated in lung and inflammatory cells in COPD and asthma (Adcock et al., 2007, 191178 ; Barnes, 2007, 191139 ). Consequently, histone deacetylase has been identified as a potential therapeutic target for epigenetic therapy (Adcock et al., 2007, 191178 ; Jones and Baylin, 2007, 191153 ). Epigenetic mechanisms have been identified as potential targets for gene-environment interactions and recent studies have demonstrated th at diet, cigarette smoking, endocrine disrupters, heavy metals and bacterial infection can alter the epigenetic profile in animals and humans (Foley et al., 2009, 191144 ). A role for PM in promoting epigenetic changes has been proposed and new studies, discussed in later chapters, provide some evidence for this pathway (Baccarelli et al., 2009, 192155 ; Liu et al., 2008, 156709 ; Reed et al., 2008, 156903 ; Tarantini et al., 2009, 192010 ; Tarantini et al., 2009, 192153 ; Yauk et al., 2008, 157164 ). Early life exposures may be especially important in this regard since periods of rapid cell division and epigenetic remodeling are likely to occur at this time (Foley et al., 2009, 191144 ; Keverne and Curley, 2008, 191154 ; Wright and Baccarelli, 2007, 191173 ). This may provide a basis for fetal origins of adult disease. It has been suggested that DNA methylation is regulated by oxygen gradients and redox status (Hitchler and Domann, 2007, 191151 ). While this is of particular importance during development where oxygen gradients and redox status are linked to cellular differentiation, these processes are also important for cell signaling during all stages of life. A common metabolic precursor for both methylation reactions and glutathione availability (involved in redox status) is methionine (Hitchler and Domann, 2007, 191151 ). Methionine availability regulates the cell’s ability to generate S-adenosyl methionine which is directly involve d in DNA and histone methylation and the cell’s ability to generate homocysteine/cysteine which is involved in glutathione biosynthesis (Hitchler and Domann, 2007, 191151 ). Furthermore, the folate cycle is a key determinant of methionine bioavailability (Hitchler and Domann, 2007, 191151 ). In this way, cellular intermediary metabolism is linked to epigenetic processes, with oxidative stress necessitating a metabolic shift resulting in decreased DNA methylation and increased glutathione production.



## 5.1.12. Lung Development

Lung development is a multi-step process which begins in embryogenesis and continues to adult life (Pinkerton and Joad, 2006, 091237 ). This allows for a long period of potential vulnerability to environmental and other stressors. Furthermore, enzymatic systems responsible for detoxification of xenobiotic compounds are not fully developed until the postnatal period (Pinkerton and Joad,

December 2009 5-112006, 091237 ). Disruption of cell signaling during develo pment could affect cellular differentiation, branching morphogenesis and overall lung growth, possibly leading to life-long consequences. Although very little is known about the effects of maternal exposure to PM on the fetus or the effects of exposure during childhood, recent animal studi es demonstrate respiratory and immune system effects of perinatal exposure to sidestream cigarette smoke (Pinkerton and Joad, 2006, 091237 ; Wang and Pinkerton, 2007, 179975 ).



# 5.2. Systemic Inflammation

Pulmonary inflammation resulting from PM e xposure may trigger systemic inflammation through the action of cytokines and other soluble mediators which leave the lung and enter the circulation ( Figure 5-5 ). Epithelial permeability may exert an important influence on this process (Section 5.1.4 ). Cytokines released by alveolar macroph ages can stimulate bone marrow production of leukocytes resulting in an increased number of total and immature leukocytes in the circulation (Van and Hogg, 2002, 088111 ; Van Eeden et al., 2001, 019018 ). They also can activate neutrophils and promote their sequestration in microvascular beds (Van Eeden et al., 2001, 019018 ). The time course of these responses varies according to th e acute or chronic nature of the PM exposure (van Eeden et al., 2005, 157086 ). Systemic inflammation is seen under conditions of mild pulmonary inflammation – and sometimes under conditions of no measurable pulmonary inflammation – following PM exposure. The time-dependent nature of pulmonary and systemic inflammatory responses may in part explain these findings since biomarkers of inflammation ar e frequently measured only at one time point. Furthermore, chronic exposures may lead to adaptive responses. In general, systemic inflammation is associated with changes in circulating white bl ood cells, the acute phase response, pro-coagulation effects, endothelial dysfunction and the development of atherosclerosis ( Figure 5-5 ). Adverse effects on the cardiovascular and cerebrovascular systems such as thrombosis, plaque rupture, MI and stroke may result. Systemic inflammation may affect othe r organ systems such as the liver or the CNS. One recent study demonstrated that alveolar macrophage-derived IL-6 mediated pro-coagulation effects in mice exposed by IT instillation to PM 10 (Mutlu et al., 2007, 121441 ). This study provides a clear link between lung cytokines and systemic responses in one model system. Whether this mechanism or others account for th e majority of extra-pulmonary effects following inhalation of PM at concentrations relevant to ambient exposures is not yet known.

December 2009 5-12

### Figure 5-5. Potential pathways for the effects of PM on the cardiovascular system.



## 5.2.1. Endothelial Dysfunction an d Altered Vasoreactivity

The lumenal surface of blood vessels is lined by endothelial cells which, in addition to providing a barrier function, are key regulators of vascular homeostasis. Endothelial cells synthesize and release vasodilators such as nitric oxide (N O) and prostacyclin and vasoconstrictors such as endothelin (ET), which act on neighboring smooth m uscle cells. ET also stimulates endothelial NO synthesis through a feedback mechanism. Inhalation of high concentrations of PM has been reported to increase ET levels in the circulation (Thomson et al., 2005, 087554 ). ET has also been proposed to play a role in hypoxia-induced MI (Caligiuri et al., 1999, 157365 ). However, the role of ET in mediating cardiovascular effects following ambien t PM exposures is unclear (Section 6.2.4.3).

December 2009 5-13Endothelial dysfunction can lead to or follow endothelial activation under conditions of systemic inflammation and/or vascular oxidative st ress. Both systemic inflammation and vascular oxidative stress have been associated with PM exposure (Nurkiewicz et al., 2006, 088611 ; Van Eeden et al., 2001, 019018 ). Cytokines activate endothelial cel ls and upregulate endothelial cell adhesion molecules. They also promote the seques tration of neutrophils in microvascular beds. Neutrophil sequestration is sometimes associated with the deposition of myeloperoxidase (MPO) on endothelial cell surfaces (Nurkiewicz et al., 2006, 088611 ). ROS-derived from neutrophils, MPO, other adhered inflammatory cells and/or other sources can perturb the balance of vasodilator and vasoconstrictor species produced by endothelial ce lls. Oxidative stress can result in decreased synthesis of NO due to limitation of the redox-sensit ive essential cofactor tetrahydrobiopterin and in decreased bioavailability of NO due to reaction with superoxide. Prostacyclin synthesis is also decreased by oxidative stress. Importantly, these pr ocesses can affect vasoreactivity such that blood vessels may be unable to respond to vasoconstrictor stimuli with compensatory vasodilation. Loss of NO and prostacyclin synthesis due to PM-dependent vascular oxidative stress may have other consequences since both exert negative in fluences on platelet and neutrophil activation. While endothelial surfaces normally are anti-throm botic, endothelial dysfunction can contribute to thrombus formation. Furthermore, inflammation an d oxidative stress associated with endothelial dysfunction can contribute to the development or pr ogression of atherosclerosis (van Eeden et al., 2005, 157086 ).



## 5.2.2. Activation of Coagulati on and Acute Phase Response

The primary function of the coagulation cascade is to stop the loss of blood after vascular injury by forming a fibrin clot. However in some cases, activation of coagulation can promote intravascular thrombosis (Karoly et al., 2007, 155890 ). It has been proposed that air pollutionassociated PM can activate clotting pathways and enhance the likelihood of an obstructive cardiac ischemic event (e.g., MI) or cerebral ev ent (e.g., stroke) (Seaton et al., 1995, 045721 ). Coagulation is regulated by intrinsic and extr insic pathways. The intrinsic pathway occurs following activation of Factor XII and does not require the addition of an exogenous agent (Mackman, 2005, 156722 ). On the other hand, the extrinsic pathway is an inducible signaling cascade that can be activated by tissue factor (TF) produced in response to inflammation or endothelial injury (Karoly et al., 2007, 155890 ). In general, platelets, red blood cells (RBCs) and endothelial cells are effector cells for inducing a pro-coagulant state in the vasculature. Circulating factors may enhance coagulation or promote activation of platelets. Cytokines formed during tissue damage and inflammation lead to TF induction. TF is the initiating stimulus for coagula tion following vascular injury or plaque erosion. Complexes of TF:Factor VIIa form on endothelial cell surfaces and play a key role in thrombin generation by initiating the extrinsic blood co agulation pathway (Gilmour et al., 2005, 087410 ). Thrombin generates fibrin from fibrinogen and ampl ifies the intrinsic pathway (Karoly et al., 2007, 155890 ). TF and thrombin also have pro-inflammato ry actions independent of coagulation functions (Chu, 2005, 155730 ); thus activation of coagulation may lead to or potentiate inflammation. Endothelial cell-derived von Willebrand factor also contributes to coagulation. The fibrinolytic system opposes these processe s by facilitating the removal of a clot. The fibrinolytic pathway is regulated by the ratio of tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI). Furthermore, the endot helial cell surface has anti-thrombotic properties due to the expression of tissue factor pathway inhibitor (TFPI) and thrombomodulin (Mackman, 2005, 156722 ). Inhibition of the fibrinolytic pathway, along w ith increased plasma viscosity and increased concentrations of plasma fibrinogen and Factor VII, contributes to a pro-thrombotic state (Gilmour et al., 2005, 087410 ). In acute lung injury, vascular cells have enhanced pro-coagulant activity and impaired fibrinolytic activity (Gilmour et al., 2005, 087410 ). In arterial atherosclerosis, TF expression is increased within plaques. As a result, spontaneous plaque rupture may trigger intravascular clotting (Karoly et al., 2007, 155890 ). Acute phase responses also play a role in hemostasis by exerting pro-coagulant effects. Cytokines such as IL-6 stimulate the liver to produce acute phase proteins including C-reactive protein (CRP), fibrinogen and antipro teases (van Eede n et al., 2005, 157086 ). To date, there is limited evidence supporting a role for ambient PM in stimulating acute phase responses (Ruckerl et al., 2007, 156931 and reviewed therein) (also Section 6.2.7 and 6.2.8 in this ISA).

December 2009 5-14

## 5.2.3. Atherosclerosis

Atherosclerosis is a chronic progressive disease which contributes greatly to cardiovascular morbidity (Libby, 2002, 192009 ). Mainly a disease of the large arteries, it is characterized by the accumulation of lipid and fibrous tissue in atheroma s, or swellings of the vessel wall (Libby, 2002, 192009 ). Although a strong link is known to exist between hypercholesterolemia and atherogenesis, there is growing appreciation of the key role play ed by inflammation in the initiation and progression of atherosclerosis (Libby, 2002, 192009 ). Furthermore, inflammation has the potential to promote thrombosis which can complicate this disorder and lead to MI and stroke (Libby, 2002, 192009 ). As discussed above, PM exposure is associated with s ystemic inflammation, potentially contributing to the development of atherosclerosis. Atheroma formation in experimental animals fe d a high fat diet begins with the accumulation of modified lipoprotein particles in the ar terial intima, as reviewed by Libby (2002, 192009 ). The modification of lipoprotein particles often invol ves oxidation. As discussed above, PM exposure is associated with oxidative stress, suggesting a potential role for PM in the modification of lipoprotein particles. Endothelial dysfunction may also be key to these early events (Halvorsen et al., 2008, 191149 ). Recent studies, which are discussed in la ter chapters, demonstrate PM-dependent endothelial dysfunction (Nurkiewicz et al., 2009, 191961 ). As described by Libby (2002, 192009 ), oxidative stress leads to lipid modification and uptake by endothelial cells and initiates an inflammatory response by activating NF- κ B. As a result, cell adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) are upregul ated and expressed in endothelial cells. Proinflammatory cytokines associated with systemic inflammation may also contribute to adhesion molecule upregulation. Subsequently, monocytes a nd T cell lymphocytes adhere to the activated endothelium, then migrate into the tunica intima directed by chemokines such as monocyte chemoattractant protein-1 (MCP-1) and IL-8. Monocytes undergo transformation to tissue macrophages and later to foam cells. As a part of this transformation, monocyte/macrophages express scavenger receptors and bind to and intern alize the modified lipoprotein particles. These cells secrete growth factors and cytokines, produce ROS, replicate within the lesion and contribute to further lesion progression. Macrophage colony-s timulating factor (M-CSF) and granulocytemacrophage colony-stimulating factor (GM-CSF) ar e thought to be involved in these latter steps which eventually lead to the formation of a fatty streak. T cell lymphocytes also become activated in the atheroma and secrete pro- or anti-inflammatory cytokines. Degranulation of mast cells found in the atheroma may also contribute to the lesion. Atheroma progression involves the proliferation of smooth muscle cells, which is stimulated by macrophage-derived growth factors (Libby, 2002, 192009 ). This results in smooth muscle accumulation in the lesion, the elaboration of extr acellular matrix material and the formation of a more bulky lesion which can occlude the arterial lume n. Matrix proteins contribute to the evolution of the lesion from a fatty streak to a fibrous plaque. Mechanisms which trigger plaque disruption, including endothelial erosions and plaque rupture, can result in thrombosis as well as in further expansion of the lesion. Resident T cells, mast cells and circulating platelets may also play a role in destabilizing plaques (Halvorsen et al., 2008, 191149 ). It is thought that most atheromatous lesions progress in a discontinuous manner due to cycles of disruption, expansion and repair. A major factor regulating plaque disruption is the thickness of the fibrous cap, with more stable plaques characterized by a thick fibrous cap (Libby, 2002, 192009 ). Collagen in the fibrous cap can be degraded by proteases, especially MMPs. Inflammation in the intima reduces collagen production by smooth muscle cells and promotes the expression and activation of MMPs. ROS may mediate MMP upregulation (Lund et al., 2009, 180257 ). Both macrophages and smooth muscle cells produce MMPs in the lesion areas (Halvorsen et al., 2008, 191149 ); fully differentiated macrophages selectively upregulate certain MMPs with more destructive potential (Newby, 2008, 191161 ). Rupture of the fibrous cap allows pro-thrombotic f actors within the plaque (e.g., TF) to come into contact with coagulation factors in the blood possi bly resulting in the formation of an occlusive blood clot. However in some cases, blood fibrinolytic mechanisms minimize clot formation and repair processes ensue. The result is a more fibrous plaque and/or an expanded lesion. The proposed role of PM in activating coagulation pathways, which was discussed above, may influence the outcome of plaque disruption. In this manner oxidative stress, inflammation and pro-coagulant activity in the blood are involved in the initiation and progression of athero matous lesions as well as plaque disruption and occlusive blood clot formation. PM exposure may c ontribute to these pathways and in fact, studies

December 2009 5-15described in later chapters demonstrate PM-depen dent effects on atherosclerosis progression (Araujo et al., 2008, 156222 ; Chen and Nadziejko, 2005, 087219 ; Sun et al., 2005, 087952 ; Ying et al., 2009, 190111 ).



# 5.3. Activation of the Autonom ic Nervous System by Pulmonary Reflexes

Chemosensitive receptors, including rapidly ad apting receptors (RARs) and sensory C-fiber receptors, are found at all levels of the respiratory tract and are sensitive to irritant particles as well as to irritant gases (Alarie, 1973, 070967 ; Coleridge and Coleridge, 1994, 156362 ; Widdicombe, 2006, 155519 ). Activation of trigeminal afferents in the nose causes CNS reflexes resulting in decreases in respiratory rate through a lengthened expi ratory phase, closure of the glottis, closure of the nares with increased nasal airflow resistance and effects on the cardiovascular system such as bradycardia, peripheral vasoconstriction and a rise in systolic arterial blood pressure (Alarie, 1973, 070967 ). Sneezing, rhinorrhea and vasodilation with subsequent nasal vascular congestion are also nasal reflex responses involving the trigeminal nerve (Sarin et al., 2006, 191166 ). Activation of vagal afferents in the tracheobronchial and alveolar re gions of the respiratory tract causes CNS reflexes resulting in bronchoconstriction, mucus secretion, mucosal vasodilation, cough, apnea followed by rapid shallow breathing and effects on the car diovascular system such as bradycardia and hypotension or hypertension (Coleridge and Coleridge, 1994, 156362 ; Widdicombe, 2003, 157145 ; 2006, 155519 ; Widdicombe and Lee, 2001, 019049 ). Some evidence suggests that cardiovascular responses may be mediated primarily by the C-fi ber receptors (Coleridge and Coleridge, 1994, 156362 ) and that irritants in the lower respiratory tract cause more pronounced cardiovascular responses than irritants in the upper respiratory tract (Widdicombe and Lee, 2001, 019049 ). Early experiments demonstrated that sectioning of the trigeminal nerve abrogated irritant effects on respiratory rate, heart rate and systolic arterial blood pressure (Alarie, 1973, 070967 ). These nasal reflexes were attributed to the ophthalmic branch of the trigeminal nerve since they were identical to reflex responses following diving or immersion of the face in water (Alarie, 1973, 070967 ). Early experiments also demonstrated that non- nasal reflexes were mediated by cholinergic parasympathetic pathways involving the vagus nerve and inhibited by atropine (Grunstein et al., 1977, 071445 ; Nadel et al., 1965, 014846 ). More recent experiments have shown that noncholinergic mechanisms may also be involved. For example, s timulation of C-fiber receptors can activate local axon reflexes. These local axon pathways are responsible for secretion of neuropeptides and the development of neurogenic inflammation (Widdicombe and Lee, 2001, 019049 ). It has been proposed that, in some cases, neurogenic pulmonary responses can switch from their normally protective function to one that perpetuate s pulmonary inflammation (Wong et al., 2003, 097707 ). Differences in respiratory tract innervation between rodents and humans suggest that C-fiber mediated neurogenic inflammation may be more impor tant in rodents than in humans (Groneberg et al., 2004, 138134 ; Widdicombe, 2003, 157145 ; Widdicombe and Lee, 2001, 019049 ) However, the role of neurogenic inflammation in mediating pulm onary responses in humans is an active area of investigation. VR1 receptors represent a subset of neuropeptide and acid-sensitive irritant receptors which belong to the transient receptor potential (TRP) family. They are located on the sensory C-fibers which lie underneath and between lung epithel ial cells and on immune and non-immune airway cells. Some investigators have focused on the role played by these receptors in mediating inflammation following exposure to PM (Veronesi and Oortgiesen, 2001, 015977 ). Exposure of bronchial epithelial cells and neurons to PM in vitro has been shown to result in an immediate increase in intracellular calcium followed by the release of neuropeptides and inflammatory cytokines (Veronesi et al., 1999, 048764 ; Veronesi et al., 2000, 017062 ). In one study, this response was found to be due to an intrinsic property of the particle core and was not metal-dependent (Oortgiesen et al., 2000, 013998 ), while in another study electrostatic charge was found to activate VR1 receptors (Veronesi et al., 2003, 094384 ). PM-mediated activation of VR1 receptors which results in increases in intracellular calcium and apoptosis in epithelial cells has also been demonstrated (Agopyan et al., 2003, 155649 ). New studies, discussed in later chapters, provide evidence for the involvement of TRPV1 irritant receptor involvement in PM-dependent responses (Ghelfi et al., 2008, 156468 ; Rhoden et al., 2005, 087878 ).

December 2009 5-16Recently it has been proposed that pulmonary reflex responses may be modulated by CNS plasticity (Bonham et al., 2006, 191140 ; Sekizawa et al., 2008, 191167 ). Plasticity is a property of neurons or synapses which allows change in response to previous events. In the case of the respiratory tract, visceral afferent inputs to the CNS are integrated primarily in the nucleus tractus soltarius (NTS) region of the brain (Bonham et al., 2006, 191140 ). Inputs from local networks, higher brain regions and circulating mediators contri bute to the reflex output (Bonham et al., 2006, 191140 ). This integration allows for plasticity in th at repeated or prolonged exposure to a particular stimuli may lead to altered reflex responses to the same or subsequent stimuli. An exaggerated reflex response was recently observed in guinea pigs expo sed to ETS for an extended period of time (Sekizawa et al., 2008, 191167 ). It is not known whether CNS plasticity influences responses following acute and chronic exposure to ambient PM, but it is a mechanism that may possibly explain hyperresponsiveness and/or adaptation of reflex-related responses. At this time, it is not clear how activation of the ANS by pulmonary reflexes contributes to the kinds of altered conduction and/or repolarization pr operties of the heart which may be linked to arrhythmias ( Figure 5-5 ). Pulmonary reflexes, as they are cu rrently understood, initially lead to increases in parasympathetic tone. However, decreased heart rate variability appears to be reflective of decreased parasympathetic tone and/or increased sympathetic tone. A PM-dependent sympathetic stress response mediated by cytokines has been postulated (Godleski et al., 2000, 000738 ), but there is little new information to support this mechan ism. Thus, activation of the autonomic nervous system by mechanisms other than pulmonary reflexes seems likely in response to PM. Very little is known about putative alternative mechanisms leading to sympathoexcitatory responses although one study demonstrated a role for the olfactory bulb-NTS pathway in regulating cardiovascular functions following smoke exposure (Moffitt et al., 2002, 191160 ). In addition, sympathoexcitatory responses occur during myocardial ischemia, mediated by the release of adenosine or the production of ROS by the myocardium (Longhurst et al., 2001, 191158 ). Hence, PM-dependent effects leading to myocardial ischemia may stimulate sympathoexcitatory responses. Furthermore, the effects of pre-existing alterations in the ANS due to disease processes (e.g., increased sympathetic tone observed in cardiac diseases) on PM responses are not understood. Possibly, integration of neural signals resulting from pulmonary and cardiac reflexes at the level of the NTS may have an influence on ANS responses to PM. Further investigation wi ll be required to clarify these mechanisms.



# 5.4. Translocation of UFPs or Soluble PM Components

UFPs can translocate across cell membrane s by non-endocytotic mechanisms involving adhesive interactions and diffusion (Geiser et al., 2005, 087362 ), as described in Section 4.3.3.1. In this study, there was no measurable loss of PM from the lung over 24 h despite the rapid translocation of inhaled UFPs into alveolar epithel ial cells and capillary endothelial cells. In another study, UFPs were localized in macrophage mito chondria as demonstrated by electron microscopy (Li et al., 2003, 042082 ). Other studies found extrapulmonary translocation of poorly soluble UFPs, but the process was slow and resulted in only a small amount leaving the lung. It is possible that in these studies PM gained access to the circulation after initial transport to the lymph nodes or the gastrointestinal system. Hence, there is limited evidence to date that UFPs or other PM size fractions access the circulation by traversing the epithelial barrier of the respiratory tract. However, soluble components from all size fractions of PM have the potential to translocate across the airway epithelium into the bronchial circ ulation or across the alveolar epithelium into the systemic circulation as depicted in Figure 5-5 . Absorption across nasal epithelium may also occur (Illum, 2006, 191205 ). Factors affecting this process include the rate of dissolution of the solute from the particle and the molecular weight of th e solute (Section 4.4). More rapid dissolution of soluble components may occur in the case of UFPs due to the higher surface/volume ratios compared with larger particles. Several interesting studies investigated the tra nslocation of water-soluble metals from the lung. Gilmour et al. (2006, 156472 ) demonstrated the rapid appearance of Zn in the plasma of rats following IT instillation of zinc sulfate (ZnSO 4 ). Similarly, Wallenborn et al. (2007, 156144 ) demonstrated the rapid appearance of water-soluble metals in the blood, heart and liver following IT instillation of oil combustion PM in rats. Using a more sensitive technique, these same investigators demonstrated the accumulation of 70 Zn, a rare isotope of Zn, in blood, heart and liver following IT

December 2009 5-17instillation of ZnSO 4 (Wallenborn et al., 2009, 191172 ). In three other studies, soluble Zn and Cu were associated with cardiac effects following IT in stillation of rats with different forms of Zn- and Cu-containing PM (Gilmour et al., 2006, 088489 ; Gottipolu et al., 2008, 191148 ; Kodavanti et al., 2008, 155907 ). These results suggest the possibility that PM-derived soluble Zn and Cu translocated across the alveolar-capillary barrier into the circulation and exerted ef fects on the heart. However, in the two studies in which barrier function was measured it was found to be compromised (Gilmour et al., 2006, 088489 ; Gottipolu et al., 2008, 191148 ) suggesting an acute lung injury response to IT instillation of high concentrations of metal. Acute lung injury is not likely to occur in healthy individuals exposed to PM at concentrations relevant to ambient levels. Cardiac effects were also observed following subchronic inhalation exposure to low concentrations of aerosolized ZnSO 4 (Wallenborn et al., 2008, 191171 ). Since it was not possible to measure extra-pulmonary Zn in this study, it remains unclear whether cardiac effects were a direct effect of translocated Zn or an indirect effect of exposure to Zn-containing PM. Nonetheless, translocation of soluble components derived from inhaled PM remains a viable hypothesis to explain some extra-pulmonary effects. Epithelial permeability is a ke y determinant of translocation and is discussed in detail in Section 4.4.2. In brief, a number of studies have measured clearance of 99m Tc-DTPA as an index of alveolar epithelial membrane integrity and permeab ility of alveolar-capillary barrier (Braude et al., 1986, 155701 ). Endothelial integrity also contributes to th e alveolar-capillary barrier and is measured by transvascular protein flux but is not discussed here (Braude et al., 1986, 155701 ). In laboratory animals, increased alveolar permeability was shown in terminally senescent mice (Tankersley et al., 2003, 096363 ). In human volunteers, epithelial permeability was transiently increased following 3 h of moderate exercise but not following 24-h exposur e to particle-rich urban air (Brauner et al., 2009, 190244 ). A previous study found that the exercise-induced increase in epithelial permeability was transient and suggested that it was due to increased ventilation and elevated vascular pressure which altered the properties of tight junctions (Hanel et al., 2003, 155826 ). Smokers (Jones et al., 1983, 155884 ) and individuals with acute respiratory distress syndrome (Braude et al., 1986, 155701 ) or interstitial lung disease (Rinderknecht et al., 1980, 191965 ) also exhibited increased alveolar epithelial permeability. The changes in smokers were reversible upon cessation of smoking. Increased airway epithelial permeability was found in as thmatics when 99m Tc-DTPA clearance was used to measure the permeability of the bronchial mucosa (Ilowite et al., 1989, 156584 ). These studies demonstrate that epithelial permeability is increased following moderate exercise and in lung syndromes associated with inflammation and suggest that compromised epithelial barrier functions in the lung may contribute to PM-mediated effects. Interaction of circulating PM or soluble PM components with vascular endothelial cells, platelets, and other leukocytes is a potential mechanism underlying the cardiovascular and systemic effects of inhaled PM. A role for PM-derived RO S and/or cellular-derived ROS has been proposed. Furthermore, soluble metals that do not redox-cycle may activate cell signaling pathways without the generation of ROS. In this way, PM may promot e adverse cardiovascular effects such as endothelial dysfunction, atherosclerosis and thrombosis. Circul ating PM or soluble PM components also have the potential to impact other organ systems. However, convincing evidence that this occurs to an appreciable extent in healthy individuals followi ng inhalation of PM at concentrations relevant to ambient exposures is lacking.



# 5.5. Disease of the Cardiovascular and Other Organ Systems

As discussed above, deposition of PM in the lung may lead not only to pulmonary disease but also to diseases of other systems ( Figure 5-5 ). In the cardiovascular system, myocardial ischemia and MI may occur as a result of the above proposed effects of PM on atherosclerosis, plaque instability, thrombosis, plaque rupture and/or altered vasoreac tivity of coronary vessels. Myocardial ischemia and MI may alter the conduction and depolarization properties of the heart and lead to arrhythmic events. In addition, thrombosis may lead to stroke and/or thromboembolic disease. Many of these processes may be interlinked and responses to ambient PM exposures may involve multiple mechanisms simultaneously with some variability depending on PM composition. Furthermore, it is not clear at this time whether PM initiates cardi ovascular disease or whether it perturbs existing disease.

December 2009 5-18In addition, recent studies which are discussed in later chapters have demonstrated PMdependent effects on the CNS (Campbell et al., 2005, 087217 ; Kleinman et al., 2008, 190074 ; Sirivelu et al., 2006, 111151 ; Veronesi et al., 2005, 087481 ; Win-Shwe et al., 2008, 190146 ). At this time, it is not known whether this is a direct or indirect consequence of PM exposure. Translocation of soluble and poorly soluble particles from the olfa ctory mucosa via the axons to the olfactory bulb of the brain has been proposed as a possible mechanism by which PM or its components may directly access the CNS. Evidence for this path way is discussed in Section 4.3.3.2. Alternative mechanisms proposed for PM-mediated CNS effect s involve systemic inflammation and autonomic responses. These are new and intriguing possibilities which warrant further investigation. PM-dependent effects on the reproductive system, reproductive outcomes and perinatal development have also been identified and are discussed in a later chapter. Mechanisms involved in these responses have not been determined. However, it seems possible that systemic inflammation and/or oxidative stress may play a role. Develo pmental windows of susceptibility may also be an important consideration. Furthermore, it has b een hypothesized that oxygen gradients and redox status are key to cell differentiation and epigenetic processes occurring during development (Section 5.1.11 ) (Hitchler and Domann, 2007, 191151 ).



# 5.6. Acute and Chronic Responses

In general, repeated acute responses may lead to cumulative effects which manifest as chronic disease. Several examples relevant to the modes of action discussed in this chapter are that of allergic responses, atherosclerosis and lung deve lopment. Allergic responses require repeated exposures to antigen over time. Co-exposure to an adjuvant, possibly DEP or ultrafine concentrated ambient particles (CAPs), can enha nce this response. Furthermore, the presence of oxidative stress, as may occur in response to PM, can contribute to allergic responses. Alle rgic sensitization often underlies allergic asthma, characterized by inflamma tion and AHR. In this way, repeated or chronic exposures involving multifactorial responses (imm une system activation, oxidative stress, inflammation) can lead to irreve rsible outcomes. Similarly, the development of atherosclerosis involves inflammation and remodeling of the blood ve ssel wall. Factors contributing to this process include systemic inflammation, endothelial dysf unction, oxidative stress and high levels of circulating lipids. PM exposure is associated with th ree out of four of these processes. The role of PM in initiating, promoting or complicating this di sease or its outcomes has yet to be determined. Critical windows of susceptibility during developm ent also provide an opportunity for repeated exposures to injurious agents to lead to irreversible changes in organ structure and function. The extended period of postnatal lung development in humans and other species heightens this vulnerability. PM may serve as such an injurious agent as has been demonstrated previously for hyperoxia (Randell et al., 1990, 191956 ). Furthermore, adverse outcomes may be precip itated by acute events superimposed on chronic disease states. In the case of allergic asthma , acute PM exposure may provoke asthmatic responses through oxidative stress and inflammatory pathways . Additionally, PM can act as a carrier of aeroallergens and other biological materials which can potentially trigger asthma attacks. Similarly, PM exposure may provoke inflammatory or thro mbotic responses leading to rupture of an atherosclerotic plaque which subsequently results in acute MI. In this way, the outcome of an acute exposure to PM may be drastically worsened by the underlying chronic disease.



# 5.7. Results of New Inhalation Studies which Contribute to Modes of Action

Prior to this review, much of the evidence for the proposed modes of action was obtained from animal studies involving IT instillation or inhalation of high concentrations of PM and from cell culture experiments. In many cases, the types of PM used were of questionable relevance to ambient exposures (i.e., high concentrations of ROFA, metals and ambient PM collected on filters). Since then, many inhalation studies have been conducte d using CAPs, combustion-derived PM, urban air

December 2009 5-19and carbon black, generally using concentrations of PM lower than 1 mg/m 3 . Much of this research has been conducted in animal models of disease. These key new studies, described in detail in Chapters 6 and 7, add to the understanding of mo des of action which are relevant to ambient PM exposure. A compilation of pertinent results is found below.

 Altered lung function including changes in respiratory frequency and AHR following short-term exposures to CAPs and com bustion-derived PM (Section 6.3.2.3)

 Mild pulmonary inflammation in response to short-term exposures to CAPs, urban air, combustion-derived PM and carbon black (Section 6.3.3.3)

 Mild pulmonary injury in response to short-term exposure to CAPs and combustionderived PM (Section 6.3.5.3)

 Inhibition of cell proliferation in the pr oximal alveolar region of neonatal animals following short-term exposure to iron-soot (Section 6.3.5.3)

 Pulmonary oxidative stress in response to short-term exposure to CAPs, urban air, combustion-derived PM, carbon black and iron-soot; pulmonary nitrosative stress in response to titanium dioxide (TiO 2 ) (Section 6.3.4.2)

 Antioxidant intervention which ameliorates PM effects on oxidative stress, allergic responses, and AHR (Sections 6.3.4.2 )

 Allergic sensitization and exacerbation of allergic responses in response to CAPs and combustion-derived PM (Section 6.3.6.3)

 Altered methylation of promoter regions of IFN- γ and IL-4 genes suggestive of proallergic Th2 gene activation following short- term exposure to combustion-derived PM in an allergy model (Section 6.3.6.3)

 Increased susceptibility to respiratory infection following exposure to combustionderived PM (Section 6.3.7.2)

 Effects on nasal epithelial mucosubstances, airway morphology and airway mucosubstances following chronic exposure to urban air-derived PM and woodsmoke (Section 7.3.5.1)

 Worsening of papain-induced emphysema following chronic exposure to urban airderived PM (Section 7.3.5.1)

 Effects on lung development following chronic exposure to urban air-derived PM (Sections 7.3.2.2 and 7.3.5.1)

 Prolonged exposure to CAPs and combustion-derived PM leading sometimes to mild pulmonary inflammation, oxidative stress and injury and sometimes to loss of inflammatory, oxidative stress and AHR respo nses which were observed after short-term exposures (Sections 7.3.2.2, 7.3.3.2, 7.3.4.1, 7.3.5.1 and 7.3.6.2)

 Hypermethylation of lung DNA following chronic exposure to combustion-derived PM (Section 7.3.5.1)

 A role for TRPV1 irritant receptors in activating local axon and CNS reflexes following short-term exposure to CAPs and combustion-derived PM (Section 6.2.9.3)

December 2009 5-20 A role for TRPV1 irritant receptors in mediating lung and heart oxidative stress through increased parasympathetic and sympathetic activity in response to CAPs (Sections 6.2.9.3 and 6.3.4.2)

 Altered heart rate variability in response to CAPs, combustion-derived PM and carbon black (Section 6.2.1.3)

 Arrhymthmic events in response to CAPs a nd combustion-derived PM (Section 6.2.2.2)

 Altered cardiac contractility following shor t-term exposure to CAPs and carbon black (Section 6.2.6.1)

 Enhanced myocardial ischemia following shor t-term exposure to CAPs (Section 6.2.3.3)

 Endothelial dysfunction and altered vascular reactivity following short-term exposure to CAPs, combustion-derived PM and TiO 2 (Section 6.2.4.3)

 Increases in blood pressure following short-term exposure to CAPs and carbon black (Section 6.2.5.3)

 Changes in blood leukocyte counts following short-term exposure to CAPs and carbon black (Section 6.2.7.3)

 Increased levels of blood coagulation factor s following short-term exposure to CAPs and on-road highway aerosols (Section 6.2.8.3)

 Systemic and cardiovascular oxidative stress in response to short-term exposure to CAPs, road dust and combustion-derived PM (Section 6.2.9.3)

 Progression of atherosclerosis, induction of TF in aortic plaques, vascular oxidative stress and altered vasomotor function following long-term exposure to CAPs in a susceptible animal model (Section 7.2.1.2)

 Vascular remodeling following chronic exposure to urban air-derived PM (Section 7.2.1.2).

 Enhanced angiotensin II-induced hypertension accompanied by vascular oxidative stress and altered vasoreactivity in response to ch ronic exposure to CAPs (Section 7.2.5.2)

 Exaggerated insulin resistance, visceral ad iposity and systemic inflammation in response to chronic exposure to CAPs and a high-fat diet (Section 7.2.3.1)

 CNS responses following short- and long-te rm exposures to CAPs and combustionderived PM (Section 6.4.3)

 Effects on the reproductive system, reproductive outcomes and developmental outcomes following chronic exposure to urban-air derived PM (Section 7.4.2)

 DNA adducts in nose, lung and liver following chronic exposure to urban air (Section 7.5.2.1)

 Germ line mutations, DNA strand breaks and global hypermethylation in sperm following chronic exposure to urban air-derived PM (Section 7.5.3)

December 2009 5-21

# 5.8. Gaps in Knowledge

The new studies highlighted in Section 5.7 confirm and extend findings from older studies. However, this increasing body of evidence does no t provide a complete picture of the biologicalpathways involved in mediating PM effects. For example, a lack of information regarding the timedependence of many responses makes it diffic ult to understand the underlying biological mechanisms. Existing gaps in knowledge include:

 The spatial distribution of retained particles in the lung and its impact

 The deposition, uptake and clearance of UFPs in the lung

 Effects of ambient PM exposures on epithelial barrier function in the lung

 Time dependence of responses

 The putative modulation of neural reflexes by pre-existing disease or other factors

 The putative role of neural reflexes beside s those involving pulmonary irritant receptors

 The putative role of ET in altering vasomotor tone following PM exposure

 The putative translocation of PM or soluble components across the epithelial barrier of the lung into the circulation

 The putative translocation of PM from olfactory epithelium to the olfactory bulb and other brain regions

Additional studies will be required to clarify th e biological mechanisms underlying the health effects of PM.

December 2009 5-22

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